Reagent combinations, media and their use in the preparation of organoids

Abstract

The present application relates to the field of cell culture, in particular to a reagent combination, a culture medium and application thereof in preparation of an organoid. The present application provides a reagent combination comprising: CHIR99021, SB431542 and LDN193189. The present application optimizes an iPSC-derived three-dimensional neuromuscular organoid generation system to overcome problems such as limited donor source, low differentiation efficiency, long-term culture required for pathological phenotype appearance, lack of standardized evaluation system, and low throughput unsuitable for large-scale drug screening, thereby providing an efficient, stable, high-throughput and clinically relevant platform for ALS disease modeling and drug development.

Description

[0001] This application claims priority to Chinese Patent Application No. 2025107882222, filed on June 12, 2025, entitled "Reagent Combination, Culture Medium and Its Application in the Preparation of Organoids", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of cell culture, and more particularly to reagent combinations, culture media, and their applications in the preparation of organoids. Background Technology

[0003] Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by the progressive degeneration and loss of upper and lower motor neurons, ultimately leading to muscle atrophy, paralysis, and death from respiratory failure. As a rare disease, ALS has a global incidence of approximately 2 cases per 100,000 people per year. In recent years, this disease has received increasing attention from society. my country has a relatively large number of ALS patients, but the average survival time from diagnosis to the end of the disease is only 2 to 5 years. Etiologically, ALS can be divided into familial and sporadic types, with sporadic cases accounting for more than 90%. However, the specific pathogenic mechanisms in this group are still unclear, posing a significant challenge to the exploration of the etiology and research on treatment.

[0004] Current treatments for ALS primarily include drug therapy, gene-targeted therapy, and stem cell therapy. However, the clinical efficacy of these therapies is very limited. FDA-approved drugs riluzole and edaravone only extend patients' survival by 3 to 6 months and do not significantly alter disease progression. Therapies targeting specific gene mutations, such as the gene therapy drug Tofersen for SOD1-ALS, are only applicable to patients carrying specific mutations. This means that over 90% of sporadic ALS patients do not benefit from current treatments, and existing treatment options fall far short of meeting the needs of patients and their families.

[0005] Treatment research for ALS faces numerous challenges, including unclear disease classification, limitations in diagnostic and assessment criteria, inadequacies of existing disease models, and a lack of more effective drug treatments. Particularly concerning disease models, while traditional animal models have played a crucial role in studying ALS mechanisms—for example, SOD1 transgenic mice can mimic some pathological features such as protein aggregation—they cannot fully reflect the complex pathology of ALS, especially the mechanisms of most sporadic cases. Furthermore, significant interspecies differences have led to the failure of many treatments that have shown efficacy in animal models in human clinical trials. This further underscores the importance of developing models that more closely resemble human pathological characteristics.

[0006] Patient-derived induced pluripotent stem cell (iPSC) models offer a patient-centered alternative research platform. By reprogramming somatic cells from familial and sporadic ALS patients to generate iPSCs, and further differentiating them into disease-related cell types, multiple disease subtypes, including those with unknown gene mutations, can be captured. These iPSC models effectively replicate patient-specific pathological features, providing crucial support for studying the pathogenesis of sporadic cases and drug screening. Although iPSC models still have certain limitations, they play a key bridging role between animal experimental data and clinical heterogeneity. This model not only provides a platform for ALS research that more closely reflects patient pathological characteristics but also opens up new possibilities for accelerating treatment development, especially in addressing the clinical needs of most sporadic cases that have not yet received sufficient attention.

[0007] In recent years, ALS research has increasingly focused on the development of three-dimensional disease models, hoping to overcome the shortcomings of two-dimensional models by establishing three-dimensional organoid systems that more closely resemble physiological conditions, thereby revealing the pathogenesis of ALS more comprehensively. These three-dimensional models can not only simulate complex intercellular interactions, but also have higher pathological realism and functionality, providing a more reliable platform for drug screening and treatment development.

[0008] However, existing iPSC-derived three-dimensional neuromuscular organoid generation systems suffer from problems such as limited donor sources, low differentiation efficiency, long-term culture required for pathological phenotype manifestation, lack of standardized evaluation systems, and low throughput, making them unsuitable for large-scale drug screening. Summary of the Invention

[0009] In view of this, the present invention provides reagent combinations, culture media, and their applications in the preparation of organoids. The present invention optimizes the iPSC-derived three-dimensional neuromuscular organoid generation system to overcome problems such as limited donor sources, low differentiation efficiency, long-term culture required for pathological phenotype manifestation, lack of standardized evaluation systems, and low throughput unsuitable for large-scale drug screening. Therefore, it provides a high-throughput, efficient, stable, and clinically relevant platform for ALS disease modeling and drug development.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides a reagent combination including: CHIR99021, SB431542 and LDN193189.

[0012] In some embodiments of the present invention, in the above reagent combination, the working concentration of CHIR99021 is 3~10µM; the working concentration of SB431542 is 3~10µM; and the working concentration of LDN193189 is 200nM~1µM.

[0013] In some embodiments of the present invention, in the above reagent combination, the working concentration of CHIR99021 is 5~10µM; the working concentration of SB431542 is 10µM; and the working concentration of LDN193189 is 200nM.

[0014] In some embodiments of the present invention, the above-mentioned reagent combination further includes: 10~50 μM Y27632.

[0015] In some embodiments of the present invention, the above reagent combination further includes: 50 μM Y27632.

[0016] The present invention also provides a culture medium comprising: the above-described reagent combination, a basal culture medium, and growth factors.

[0017] In some embodiments of the present invention, the basal culture medium mentioned above includes: Neurobasal medium and DMEM / F12 medium containing supplements and antibiotics;

[0018] The supplements include: N2 supplements and B27 supplements;

[0019] The antibiotics include: penicillin-streptomycin;

[0020] The growth factors include one or more of bFGF, HGF, and IGF.

[0021] In some embodiments of the present invention, the supplement in the above-mentioned culture medium further includes: Glutamax and / or ascorbic acid.

[0022] In some embodiments of the present invention, the working concentrations of the growth factors in the culture medium are: 10-40 ng / mL of bFGF, 1-10 ng / mL of HGF, and 1-10 ng / mL of IGF.

[0023] In some embodiments of the present invention, the working concentrations of the growth factors in the culture medium are: 10-40 ng / mL of bFGF, 2 ng / mL of HGF, and 2 ng / mL of IGF.

[0024] The present invention also provides the application of the above-described reagent combination and / or the above-described culture medium in the preparation of neuromuscular organoids.

[0025] The present invention also provides the application of the above-described reagent combination and / or the above-described culture medium in the preparation of neural mesodermal progenitor cells.

[0026] This invention also provides a method for preparing neuromuscular organoids, comprising the following steps:

[0027] S1: After seeding induced pluripotent stem cells, induce differentiation culture for 1 day, culture with double inhibition for 1.5 days, and then transfer to a new culture vector;

[0028] S2: Culture the cells that have completed differentiation in S1, passage them, expand the culture, and obtain the neuromuscular organoids;

[0029] The induction described in S1 uses the basal culture medium, the CHIR99021, and the bFGF from the above-mentioned culture medium;

[0030] The dual inhibition described in S1 uses the basal culture medium, CHIR99021, bFGF, SB431542, and LDN193189 from the above-mentioned culture media.

[0031] In some embodiments of the present invention, the concentration of CHIR99021 in the induction process of the above preparation method is 5 µM; and the concentration of CHIR99021 in the dual inhibition process is 10 µM.

[0032] In some embodiments of the present invention, the novel culture carrier described in the preparation method S1 above includes: the Aggrewell system.

[0033] In some embodiments of the present invention, the cell culture in the above-mentioned preparation method S2 is an organoid formation stage, specifically including the following steps:

[0034] S1: Day 1 to Day 3 after transfer to the new culture medium: partial removal and replenishment of the culture medium; the culture medium includes: the basal culture medium, 10 ng / mL bFGF, 2 ng / mL HGF and 2 ng / mL IGF;

[0035] S2: On day 4, the generated organoids were transferred from the Aggrewell system to 60 mm culture dishes.

[0036] In some embodiments of the present invention, the passage in the preparation method S2 described above is: organoid expansion stage, specifically including the following steps: on the 6th to 8th day after the transfer to the new culture medium, the culture medium is partially removed and replenished; the culture medium is the basal culture medium.

[0037] In some embodiments of the present invention, the specific steps of the expanded culture in the preparation method S2 above include: transferring to a 100 mm culture dish on the 10th day after the transfer to the new culture carrier, and adding the basal culture medium.

[0038] The present invention also provides neuromuscular organoids obtained by the above preparation method.

[0039] The present invention also provides the application of the above-mentioned neuromuscular organoids in any of the following:

[0040] (a) Constructing an organoid model of amyotrophic lateral sclerosis (ALS);

[0041] (b) Constructing drug models for screening and / or evaluating amyotrophic lateral sclerosis (ALS);

[0042] (c) Screening for drugs for amyotrophic lateral sclerosis (ALS);

[0043] (d) Treatment of amyotrophic lateral sclerosis (ALS);

[0044] (e) Prepare products for the treatment of amyotrophic lateral sclerosis (ALS).

[0045] The beneficial effects of this invention include:

[0046] (1) Significantly improved differentiation efficiency: In previous differentiation systems, the differentiation efficiency of neuroectoderm and mesodermal progenitor cells was low, and the cell death rate was high. This invention significantly improves differentiation efficiency and reduces cell death rate by introducing a "dual SMAD inhibition" strategy and optimizing the stepwise concentration adjustment of CHIR99021. This improvement makes the cell differentiation process more efficient and stable, providing a solid technical foundation for organoid generation.

[0047] (2) High-throughput organoid generation: Previous organoid generation systems have shortcomings in terms of throughput and uniformity. This invention employs the Aggrewell system, where each well is designed to contain up to 1,200 microwells for organoid generation. Each well can simultaneously generate up to 1,200 organoids, and the organoids exhibit greater consistency in morphology and cellular composition. This technology significantly improves production efficiency and uniformity, meeting the needs of large-scale experiments and providing reliable technical support for drug screening and disease research.

[0048] (3) Comprehensive functional assessment methods: The assessment of the optimized system is relatively comprehensive. At the structural level, immunofluorescence staining confirmed the formation of neurons, myofibrils and complete neuromuscular junctions. At the functional level, experiments showed that muscle contraction can be effectively regulated by neurons. At the omics level, multi-omics sequencing verified that the cellular composition of the optimized system remained highly stable. At the molecular level, molecular benchmark analysis further confirmed that the system significantly improved the early induction efficiency and ensured the high fidelity of cell differentiation trajectory.

[0049] (4) Improved reliability of disease models: Traditional functional assessment methods for ALS disease models are relatively simple (mostly based on information about neuronal degeneration), making it difficult to fully reveal the complexity of the disease phenotype. This invention develops a systematic functional assessment method for neuromuscular junctions in ALS, covering comprehensive detection of multiple aspects such as muscle contraction ability, number of neuromuscular junctions, neuronal apoptosis, autophagy, stress granule formation, and TDP43 protein abnormalities. Currently, this system has been validated on induced pluripotent stem cells derived from familial ALS patients and sporadic ALS patients carrying multiple gene mutations such as C9orf72, FUS, and TDP-43. This comprehensive assessment system can accurately reflect the functional state of organoids, providing reliable experimental evidence for disease mechanism research and drug screening.

[0050] (5) Advantages in drug screening applications: Drug screening requires standardized models and reliable data support. Utilizing the optimized organoid model and standardized functional assessment method of this invention, the intervention effect of drugs on disease phenotypes can be accurately quantified. This technology significantly improves the efficiency and accuracy of drug screening, providing strong technical support for drug development in the treatment of neuromuscular diseases. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0052] Figure 1 Demonstrate the experimental procedure;

[0053] Figure 2 The study showed that dual SMAD inhibition significantly enhanced the specific differentiation of neuroectodermal lineages; where: A represents a quantitative comparison of organoid morphological characteristics on day 5: the original organoid culture (Previous protocol) and the culture transferred to Aggrewell (Move to...). Analysis of organoid elongation after Aggrewell plate transfer, with each data point representing an independent differentiation experiment. The results showed that organoid elongation was significantly reduced after transfer to the Aggrewell plate. B and C show the immunofluorescence staining (B) and quantitative analysis (C) of neuroectodermal (SOX1) and mesodermal (PAX3) markers before and after transfer to the Aggrewell plate on day 5 of differentiation. The results showed that the cellular expression levels of SOX1 and PAX3 markers were lower after transfer to the Aggrewell plate. D is a schematic diagram of the experimental workflow for optimizing the differentiation strategy. E shows the immunofluorescence staining results of SOX1 and PAX3 markers in each treatment group on day 5 of differentiation, including the control group (Aggrewell_Control), the double SMAD inhibition group (Aggrewell_2SMADi), the SB431542-only treatment group (Aggrewell_SB431542), and the LDN193189-only treatment group (Aggrewell_LDN193189). F shows the quantitative analysis of the expression level of the neuroectodermal marker SOX1 on day 5 of differentiation. The results showed that combined inhibition of dual SMAD significantly upregulated SOX1 positive signal;

[0054] Figure 3 This study demonstrates that CHIR99021 promotes NMP differentiation towards mesodermal progenitor cells by activating the Wnt signaling pathway. A shows a schematic diagram of the experimental procedure; B shows morphological observations of NMP cells under different CHIR99021 concentrations, including 3 µM, 5 µM, 10 µM, stepwise concentrations (days -2.5 to -1.5: 5 µM; day -1.5 to 0: 10 µM), and reverse stepwise concentrations (days -2.5 to -1.5: 10 µM; day -1.5 to 0: 5 µM). C shows the immunofluorescence staining results on day 5 of differentiation, indicating the expression of mesodermal marker PAX3 (pink) and neuroectodermal marker SOX1 (yellow); D shows the immunofluorescence staining results on day 20 of differentiation, indicating the expression of neural marker TUJ1 (yellow) and muscle marker MyHC (cyan); E shows the quantitative analysis results of immunofluorescence staining on day 5 of differentiation, showing that the proportion of PAX3 positive cells increased significantly under higher concentrations of CHIR99021; F shows the quantitative analysis results of immunofluorescence staining on day 20 of differentiation, showing that the proportion of MyHC positive cells was significantly upregulated under higher concentrations of CHIR99021 treatment.

[0055] Figure 4This document describes the construction and functional evaluation of a high-throughput organoid model. A shows a schematic diagram of the differentiation optimization strategy for constructing the high-throughput organoid model, addressing the low throughput and uniformity issues of previous differentiation methods. B shows the immunofluorescence staining results of the optimized neuromuscular organoid (OptiNMO) on day 50 after differentiation. The upper image is a schematic diagram of the functional NMJ unit cell composition, and the lower image shows TUJ1⁺ neurons projecting neurites into an orderly arranged skeletal muscle compartment (MyHC). The TUJ1⁺ neurites are in contact with the α-BTX-positive (αBTX) acetylcholine receptor (AChR) clusters on the surface of MyHC⁺ muscle fibers, and S100β⁺ terminal Schwann cells cover the nerve endings at the αBTX⁺ sites, consistent with the typical structural characteristics of a functional neuromuscular junction (NMJ). C shows the OptiNMO muscle contraction function detection process. D shows the functional experimental results of the neuromuscular organoid: glutamate (50 μM) and tetrodotoxin (TTX) were applied, respectively. After 3 and 10 minutes of treatment with 1 μM, muscle contraction was observed to exhibit both promoting and inhibiting effects, thus confirming that the contractile activity of OptiNMO is regulated by the nervous system.

[0056] Figure 5 The integrated analysis of single-cell nuclear transcriptome (snRNA-seq) and chromatin accessibility (snATAC-seq) was performed. Multi-omics integrated analysis was conducted on RefNMO and OptiNMO samples at multiple key time points from NMP to D3 to D50. The results of multi-omics joint clustering and cell type annotation showed that the cellular composition of organoids before and after optimization was highly consistent, confirming that the differentiation optimization strategy did not change the basic cellular structure of organoids.

[0057] Figure 6 The stacked bar charts visually present the co-embedding integrated dataset, and the relative abundance distribution of annotated cell types at each differentiation stage in snRNA-seq and snATAC-seq. A systematic cross-sectional comparison of RefNMO and OptiNMO further confirms that this differentiation optimization strategy improves functional characteristics without disturbing the basic cellular composition inherent in organoids.

[0058] Figure 7 Immunofluorescence staining images showing the integrity of the RefNMO neuromuscular junction (NMJ) on day 50 of differentiation; the figures show representative results from five independent donors (Donor 1, 19, 7, 18 and 30) to assess the formation and integrity of the NMJ structure in the optimized system;

[0059] Figure 8The RefNMO muscle contraction time trajectory is shown on day 50 after differentiation; AE corresponds to the contraction records of Donor 1, 19, 7, 18 and 30 samples respectively. The vertical axis is the area below the threshold and the horizontal axis is the number of frames acquired, showing the contraction activity dynamics and phenotypic differences of organoids from different donors.

[0060] Figure 9 Immunofluorescence staining results of the integrity of OptiNMO neuromuscular junction (NMJ) on day 50 of differentiation under optimized conditions are shown; the figure shows representative images of five independent donors (Donor 1, 19, 7, 18 and 30) to assess the formation and integrity of NMJ structures in the optimized system.

[0061] Figure 10 The OptiNMO muscle contraction time-series trajectory is shown on day 50 after differentiation under optimized conditions; AE corresponds to the contraction records of Donor1, 19, 7, 18 and 30 samples, respectively. The vertical axis is the area below the threshold, and the horizontal axis is the frame number, showing the contractile functional characteristics of each donor organoid in the optimized system.

[0062] Figure 11 This study presents a functional comparative analysis of the two differentiation systems, RefNMO and OptiNMO. Specifically: A) is a quantitative analysis of muscle contraction capacity (contraction counts / 3 min) based on organoids from five independent donors, showing that the contraction frequency of OptiNMO is significantly higher than that of RefNMO; B) is a quantitative immunofluorescence statistical analysis of NMJ integrity based on five independent donors, showing that the optimized OptiNMO significantly improves the neuromuscular junction integrity compared to the original method.

[0063] Figure 12 The results show that OptiNMO significantly improves the differentiation uniformity and stability across donors and batches. Specifically: A shows representative bright-field images of OptiNMO from multiple independent donors on day 50 of differentiation, demonstrating highly consistent organoid morphology and excellent differentiation uniformity; B shows a quantitative analysis of the OptiNMO differentiation success rate. The results indicate that the optimized system maintains highly stable differentiation efficiency across different donor sources and independent experimental batches, significantly overcoming batch fluctuations and donor-to-donor differences common in traditional methods. This confirms that the OptiNMO platform possesses excellent reproducibility and potential for standardized application.

[0064] Figure 13This study demonstrates how OptiNMO recreates neuromuscular dysfunction and TDP-43 pathological features associated with ALS. Specifically: A is a schematic diagram of the study design, comparing organoids derived from healthy controls and ALS patients differentiated using the OptiNMO platform (≥3 healthy donors and ≥6 ALS donors were included); B is a longitudinal quantitative analysis of the density of neurally innervated neuromuscular junctions (NMJs) in OptiNMO, normalized to MyHC⁺ myofiber area by the number of neurally innervated αBTX⁺ synaptic clusters, showing significant loss of synaptic structure in the ALS group during culture; C is the spontaneous contraction frequency of OptiNMO (times / 3). Longitudinal tracking quantification (min) showed that contractile function in the ALS group progressively declined; DF assessed neuronal apoptosis (marker: CC3), autophagy (marker: P62), and stress granule formation (marker: G3BP), with results showing significant increases in all these indicators in the disease group; GJ analyzed the pathological evolution and mislocalization of TDP-43; G is a schematic diagram of TDP-43 pathological progression, showing the cascade evolution from normal nuclear localization to pathological cytoplasmic mislocalization / aggregation and neuronal degeneration; H represents differentiation. Representative immunofluorescence images of TUJ1-positive neurons in OptiNMO on day 80 visually present the abnormal cytoplasmic aggregation phenotype of TDP-43 and phosphorylated TDP-43 (pTDP-43) in the ALS group; IJ is a quantitative analysis of the ratio of TDP-43 nucleoplasmic fluorescence intensity on day 50 and day 80 (I) and the proportion of cytoplasmic pTDP-43 neurons (J) (each data point represents an independent organoid), confirming that OptiNMO can effectively simulate the progressive pathological features of TDP-43 protein abnormality in the ALS process.

[0065] Figure 14 This shows a flowchart of the drug screening process;

[0066] Figure 15 A small molecule drug was screened out as a potential candidate drug. During the 20-day treatment period starting on day 50, the drug significantly enhanced muscle contraction strength and improved the integrity of the neuromuscular junction (NMJ). Its repair effect was dose-dependent. More importantly, this efficacy was replicated in cell lines with different backgrounds, including SOD1 mutation, C9orf72 mutation and sporadic ALS cell lines.

[0067] Figure 16This study demonstrates the effectiveness of a drug screening platform for neuromuscular organoids using motor neurons. A shows the experimental steps for drug administration to motor neurons; B shows the identification of motor neuron biomarkers; C shows the phenotype of neurofilament length in motor neurons; D shows the changes in neurofilament length after drug administration (where X01 is the screened drug, and ROPI and RILU are positive drugs for treating ALS and can be used as positive controls); E shows TUJ1 immunofluorescence staining of cells 5 days after drug treatment, revealing changes in neurofilament phenotype. Images include neurons derived from healthy individuals (HC), sporadic ALS patients (sALS-1), and treatment groups treated with 10 µM X01, 40 µM X01, and positive drug controls (RILU and ROPI), respectively. ROPI refers to Ropinirole Hydrochloride, and RILU refers to Riluzole. Detailed Implementation

[0068] This invention discloses reagent combinations, culture media, and their application in the preparation of organoids.

[0069] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0070] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0071] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0072] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0073] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0074] This invention optimizes the iPSC-derived three-dimensional neuromuscular organoid generation system to overcome problems such as limited donor sources, low differentiation efficiency, long-term culture required for pathological phenotype manifestation, lack of standardized evaluation systems, and low throughput unsuitable for large-scale drug screening. Therefore, it provides a high-throughput, efficient, stable, and clinically relevant platform for ALS disease modeling and drug development. Specific objectives include:

[0075] 1. Establish models with diverse donor sources to enrich genetic background diversity.

[0076] This invention utilizes iPSCs derived from healthy donors and patients with different subtypes of ALS (including familial and sporadic) to differentiate into NMOs. By combining single-cell transcriptomics and single-cell open chromatin analysis (ATAC-seq), it reveals the differences in cellular components and proportions among patients, aiming to uncover pathology-related molecular characteristics.

[0077] 2. Optimize differentiation systems to improve efficiency and accelerate the emergence of pathological phenotypes.

[0078] This invention introduces Aggrewell high-throughput culture technology to replace the previous 96-well plate differentiation system, significantly increasing the throughput of organoid production. With this optimized system, pathological features (such as apoptosis, stress granule formation, and protein aggregation) can be observed on day 50 (D50), significantly reducing the time required by previous methods and thus greatly improving the efficiency of drug screening.

[0079] 3. Improve differentiation strategies to achieve cell lineage developmental balance

[0080] This invention employs a Dual-SMAD inhibition technique and a stepwise concentration-controlled CHIR99021 strategy to precisely regulate the WNT, BMP, and TGFβ signaling pathways, ensuring a balance in the differentiation efficiency of neuroectodermal and mesodermal cells. The generated NMOs can not only form functional NMJs but also mimic key pathological features of ALS, such as impaired muscle contraction, neuronal degeneration, and pathological protein aggregation.

[0081] 4. Establish a standardized screening system to support drug development.

[0082] This invention establishes a stable and reproducible drug screening platform by unifying differentiation procedures, phenotypic analysis, and functional validation standards. This platform significantly reduces experimental bias, improves the accuracy of drug screening results, and provides reliable support for the development of ALS-related drugs. It also accelerates research into pathological mechanisms and the development of treatments.

[0083] In summary, this invention establishes an efficient, high-throughput, and standardized platform by optimizing the iPSC-derived three-dimensional neuromuscular organoid generation system, thereby overcoming the shortcomings of existing technologies and fully supporting ALS disease mechanism research and drug development.

[0084] The prior differentiation protocol or original system of this invention is the method of generation of neuromuscular organoids in 3D in Faustino Martins, Jorge-Miguel et al. “Self-Organizing 3D Human Trunk Neuromuscular Organoids.” Cell stem cellvol. 26,2 (2020): 172-186.e6. doi:10.1016 / j.stem.2019.12.007.

[0085] In Examples 1 to 4 and Verification Examples 1 to 3 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0086] The present invention will be further illustrated below with reference to the embodiments:

[0087] Example 1

[0088] I. Induction and Differentiation Stage of Neuromesodermal Progenitors (NMPs)

[0089] D-2.5~D-1.5: Cell digestion and seeding

[0090] Induced pluripotent stem cells (iPSCs) obtained by reprogramming PBMCs from the blood of healthy individuals or patients using Sendai virus were used for cell digestion and plating. First, the cells were digested using an appropriate amount of TrypLE Express enzyme. The culture dish was gently agitated to ensure the digestion solution evenly covered the cell surface. Cells were observed under a microscope until they gradually detached from the bottom of the dish. Then, N2B27 medium (N2B27 basal medium is a mixture of 1% (v / v) N2 supplement, 2% (v / v) B27 supplement, 1% (v / v) L-glutamine substitute Glutamax, 200 nM L-ascorbic acid, and 1% (v / v) penicillin-streptomycin; the mixture consists of a 1:1 volume ratio of Neurobasal medium and DMEM / F12 medium) was added to terminate the digestion. The cell suspension was collected from the culture dish, centrifuged, the supernatant was discarded, and the cells were resuspended in fresh culture medium. Cell density was calculated using a cell counter or hemocytometer, and the cell concentration was adjusted to 40,000 cells / cm². Cells were then evenly distributed into pre-coated Matrigel-coated 6-well plates. The following NMP induction medium (containing N2B27 medium, 5 µM CHIR99021 (GSK-3β inhibitor), and 40 ng / mL bFGF) was added. Finally, the cells were incubated overnight at 37°C with 5% CO2 to ensure cell adhesion and differentiation.

[0091] D-1.5~D0: Culture medium replacement

[0092] Gently aspirate and discard the original culture medium, being careful not to aspirate the cells. Add fresh culture medium, N2B27 medium (containing 10 μM CHIR99021, 40 ng / mL bFGF, 200 nM LDN193189, and 10 μM SB431542). Return the cell culture plate to the incubator and continue culturing and differentiation.

[0093] D0: Culture medium change and cell transfer to the Aggrewell system

[0094] At day 0, discard the original culture medium and add fresh N2B27 medium (containing 10 ng / mL bFGF, 2 ng / mL HGF, 2 ng / mL IGF, and 50 μM Y27632). Prepare the Aggrewell 400 culture system, ensuring it is clean and pretreated (e.g., coated or moistened). Count the cell suspension collected from the 6-well plates, ensuring 1000 cells are added to each well. Gently add the cell suspension to the Aggrewell wells, ensuring even distribution of cells. Finally, incubate the Aggrewell system in an incubator and observe cell aggregation to form organoids.

[0095] II. NMO organoid formation stage

[0096] NMO D1: Partial removal and replenishment of culture medium

[0097] On day 1 (D1) of NMO generation, gently remove 1.5 mL of culture medium using a pipette, taking care to avoid aspirating the organoids or disturbing their structure. Then, add 1 mL of fresh culture medium, N2B27 medium (containing 10 ng / mL bFGF, 2 ng / mL HGF, and 2 ng / mL IGF). Return the Aggrewell system to the incubator and continue incubating the organoids.

[0098] NMO D2: Partial removal and replenishment of culture medium

[0099] On the second day after NMO generation, gently remove 1 mL of culture medium and add 1 mL of fresh culture medium, using the same composition as NMO D1.

[0100] NMO D3: Partial Removal and Replenishment of Culture Medium

[0101] On day 3 of NMO generation, gently remove 1 mL of culture medium and replenish with 1 mL of culture medium containing the same components as NMO D1. Handle with care to avoid damaging the organoid structure.

[0102] NMO D4: Organoid transfer to new culture dishes

[0103] On day 4 of NMO generation, the generated organoids were transferred from the Aggrewell system to 60 mm culture dishes (Corning). The organoids were gently handled with a pipette to avoid mechanical damage and to ensure that they were evenly distributed in the culture dish.

[0104] III. Organoid Expansion Stage

[0105] NMO D6: Partial Removal and Replenishment of Culture Medium

[0106] On day 6 of NMO generation, half of the culture medium was removed and an equal volume of fresh N2B27 medium was added (N2B27 basal medium is a mixture of 1% (v / v) N2 supplement, 2% (v / v) B27 supplement, 1% (v / v) L-glutamine substitute Glutamax, 200 nM L-ascorbic acid and 1% (v / v) penicillin-streptomycin; the mixture consists of Neurobasal medium and DMEM / F12 medium in a 1:1 volume ratio) to ensure sufficient volume of medium to cover the organoids and support their continued development.

[0107] NMO D8: Partial Removal and Replenishment of Culture Medium

[0108] On day 8 of NMO generation, repeat the above procedure, remove half of the culture medium, and replenish with an equal amount of fresh N2B27 medium (N2B27 basal medium is a mixed medium containing 1% (v / v) N2 supplement, 2% (v / v) B27 supplement, 1% (v / v) L-glutamine substitute Glutamax, 200 nM L-ascorbic acid, and 1% (v / v) penicillin-streptomycin; the mixed medium consists of Neurobasal medium and DMEM / F12 medium in a 1:1 volume ratio).

[0109] NMO D10: Organoid transfer to larger culture dishes

[0110] On day 10 of NMO generation, organoids were transferred to 100 mm culture dishes (Corning). 15 mL of N2B27 medium (same as NMO D8) was added to each dish to ensure that the organoids had sufficient space for further development.

[0111] NMO D10~D30: Change the culture medium regularly.

[0112] Starting from day 15, change the culture medium every 5 days. Use fresh N2B27 medium (same as NMO D8) to ensure that the organoids receive continuous nutritional support.

[0113] NMO D30: Organoid Transfer and Culture Medium Replacement

[0114] On day 30, the organoids were transferred again to new 100 mm culture dishes (Corning) and 15 mL of N2B27 medium (same as NMO D8) was added. Starting from day 30, the medium was changed every 10 days.

[0115] IV. Long-term cultivation and maintenance phase

[0116] NMO D40~D80: Change the culture medium regularly.

[0117] Change the culture medium every 10 days between days 40 and 80. Use fresh N2B27 medium to ensure the organoids are always under suitable culture conditions.

[0118] Example 2

[0119] Despite some progress in the field of neuromuscular organoids (NMOs) in recent years, current NMO systems still have many limitations, such as limited donor diversity, low throughput, and a lack of standardized pharmacological and preclinical screening indicators. To address these issues, we focus on optimizing differentiation systems for generating NMOs from induced pluripotent stem cells (iPSCs), aiming to improve experimental scalability and enhance overall efficiency.

[0120] We transitioned our culture platform from the previous neuromuscular junction organoid differentiation process to the Aggrewell system, enabling the simultaneous production of up to 1,200 organoids per well, significantly improving the scalability of our experiments. However, organoids cultured in the Aggrewell system using the previous differentiation protocol exhibited unsatisfactory results by day 5 of differentiation, including incomplete organoid structure formation and significantly reduced expression of the mesodermal marker PAX3 and the neuroectodermal marker SOX1 (e.g., Figure 2 As shown in AC).

[0121] Example 3: Bilateral SMAD inhibition significantly enhanced the specific differentiation of neuroectodermal lineages in neuromuscular junction organoids.

[0122] We introduced a temporary "dual-SMAD inhibition" strategy (addition of 10 μM SB431542 and 200 nM LDN193189) during the early NMO induction phase. With this intervention, we observed a significant upregulation of neuroectodermal progenitor markers on day 5 of differentiation. Specifically, we analyzed data from four donors: Donor_1, 13, 6, and 19. Immunofluorescence (IF) staining showed that dual-SMAD inhibition significantly increased the expression level of the neuroectodermal progenitor marker (SOX1), showing a significant increase compared to the original conditional control group without dual-SMAD inhibition. This enhancing effect was consistent across all donor samples, indicating that the dual-SMAD inhibition strategy has a reliable role in improving progenitor differentiation (e.g., ...). Figure 2 (as shown in DF).

[0123] Table 1

[0124]

[0125] Example 4: CHIR99021 promotes NMP differentiation towards mesodermal progenitor cells by activating the Wnt signaling pathway.

[0126] Table 2

[0127]

[0128] To further optimize WNT signaling pathway activation, we tested different concentrations of CHIR99021 (3 µM, 5 µM, and 10 µM). Although higher concentrations (10 µM) significantly upregulated the expression of mesodermal progenitor cell markers, they also led to increased cell death in the early NMP stage. To address this issue, we employed two stepwise concentration strategies: the first used 5 µM on day 1 and 10 µM on day 2; the second used 10 µM on day 1 and 5 µM on day 2, to investigate whether these adjustments affected differentiation outcomes. Experimental results showed that the concentration used on day 1 had a more significant impact on NMP differentiation outcomes (e.g., ...). Figure 3 (As shown in B).

[0129] Subsequently, on day 5 of differentiation, we used immunofluorescence to detect the expression distribution of the neuroectodermal marker SOX1 and the mesodermal marker PAX3. Quantitative analysis confirmed that a time-incremental strategy of CHIR99021 concentration significantly upregulated the proportion of PAX3-positive cells, driving the differentiation lineage towards mesodermal bias (e.g., ...). Figure 3 (As shown in C and E). By day 20 of differentiation, immunofluorescence results showed that the neural marker TUJ1 and the myogenic marker MyHC were expressed in a coordinated manner, further validating that this optimization strategy effectively improved the differentiation output of the mesodermal lineage while maintaining the developmental balance of the neuromuscular dual lineage (e.g., as shown in C and E). Figure 3 (As shown in D and F).

[0130] Based on these findings, we adopted a stepwise concentration strategy as an optimized approach for high-throughput iPSC-NMO differentiation in the Aggrewell system. This method not only effectively reduced cell death but also significantly increased the expression of the mesodermal progenitor cell marker PAX3, thereby ensuring an efficient and scalable differentiation process.

[0131] Verification Example 1

[0132] Through these optimizations, we successfully established a standardized, efficient, and high-throughput organoid culture protocol. Functional assessments showed that OptiNMO from healthy donors not only formed a structurally intact neuromuscular junction (NMJ) but also exhibited rhythmic contractile function regulated by neurons. Multi-omics integrated analysis of single-cell nuclear transcriptomics (snRNA-seq) and chromatin accessibility (snATAC-seq) further confirmed that the cell type composition of the organoids before and after optimization was highly consistent, indicating that this strategy promoted functional maturation while completely preserving the basic cellular structure (such as...). Figure 4 , Figure 5 and Figure 6 (As shown). Compared with the original system, OptiNMO significantly enhances muscle contraction function, and both NMJ synapse density and contraction kinetics parameters are greatly improved (e.g. Figures 7-11 (As shown). Furthermore, this optimized platform exhibits excellent differentiation uniformity and stability across different donors and independent batches (e.g., Figure 12 As shown in the figure, this lays a solid foundation for its standardized preparation and transformation application.

[0133] Verification Example 2

[0134] Based on this optimized differentiation system, we successfully constructed neuromuscular organoids derived from ALS patients and systematically validated their ability to reproduce the core pathological features of ALS. This optimized platform stably induced donor-specific disease phenotypes while maintaining high throughput and structural homogeneity. Compared with healthy controls, patient-derived organoids exhibited significant functional decline and structural degeneration: significantly reduced muscle contractility and markedly decreased neuromuscular junction (NMJ) density. At the cellular and molecular levels, the neuronal regions of patient organoids showed increased apoptosis levels, impaired autophagy, and significantly upregulated expression of typical ALS pathological markers such as pathological cytoplasmic mislocalization / aggregation of TDP-43 protein (e.g., increased apoptosis levels, impaired autophagy, and significantly upregulated expression of typical ALS pathological markers such as TDP-43 protein pathological cytoplasmic mislocalization / aggregation). Figure 13 (As shown). The above multidimensional phenotypic data collectively confirm that the OptiNMO platform can faithfully simulate the cascade characteristics of neuromuscular degeneration in the progression of ALS, providing a reliable in vitro model for disease mechanism analysis and high-throughput drug screening.

[0135] Verification Example 3

[0136] Neuromuscular organoids generated in optimized differentiation systems offer significant advantages, making them ideal models for drug screening. These organoids exhibit high throughput, structural homogeneity, and characteristic phenotypes of ALS, along with higher differentiation maturity, making them suitable for early-stage drug screening. We have conducted preliminary drug screening studies (e.g., Figure 14 (As shown)

[0137] We selected induced pluripotent stem cells (i.e., those from patients with sporadic ALS, C9orf72 mutations, and SOD1 mutations) from patients with different ALS disease backgrounds, differentiated them to generate neuromuscular junction organoids, and conducted drug screening experiments. Initial screening was based on assessing the effects of drugs on the integrity of the neuromuscular junction and the recovery of muscle contraction frequency and number of repetitions. These functional parameters were quantitatively measured using automated video analysis and α-BTX staining. The experiment was designed to add different types of small molecule drugs to organoids differentiated to day 50, and after 20 days of treatment, the neuromuscular junction structure and muscle contraction function were scored. Finally, we screened a candidate drug, X01 (e.g., ...), from the small molecule drugs. Figure 15 (as shown in the figure), and further evaluated the effects of the drug on organoids at different concentrations. The results showed that using 10 µM of this small molecule drug could significantly improve the disease phenotype.

[0138] Furthermore, we validated the efficacy of this drug in other ALS models, such as motor neuron models. Using induced pluripotent stem cells from different ALS disease backgrounds differentiated into motor neurons, 10 µM and 40 µM of the small molecule drug were added at day 25 of differentiation. The results showed that the drug significantly improved the recovery of the neurite length phenotype, demonstrating good efficacy (e.g., Figure 16 (As shown in the figure). This result demonstrates that our developed drug screening platform has high reliability and effectiveness, providing important support for ALS disease mechanism research and drug development.

[0139] Including flowchart presentation (e.g.) Figure 14 (as shown) and preliminary efficacy evaluation of the screened small molecule drugs (such as...) Figure 15 (As shown). In the future, we plan to further expand the types of drugs screened, including small molecule drugs, gene-targeted drugs, and cell therapy drugs, to further enhance the potential of neuromuscular junction organoids in ALS disease research and application.

[0140] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reagent combination, characterized in that, include: CHIR99021, SB431542 and LDN193189.

2. The reagent combination as described in claim 1, characterized in that, The working concentration of CHIR99021 is 3~10µM; the working concentration of SB431542 is 3~10µM; and the working concentration of LDN193189 is 200nM~1µM.

3. The reagent combination as described in claim 1 or 2, characterized in that, Also includes: 10~50 μM Y27632.

4. A culture medium, characterized in that, include: The reagent combination, basal culture medium, and growth factor as described in any one of claims 1 to 3.

5. The culture medium as described in claim 4, characterized in that, The basal culture media include Neurobasal medium and DMEM / F12 medium containing supplements and antibiotics; The supplements include: N2 supplements and B27 supplements; The antibiotics include: penicillin-streptomycin; The growth factors include one or more of bFGF, HGF, and IGF. Preferably, the supplement also includes Glutamax and / or ascorbic acid.

6. The culture medium as described in claim 5, characterized in that, The working concentrations of the growth factors are: 10-40 ng / mL of bFGF, 1-10 ng / mL of HGF, and 1-10 ng / mL of IGF.

7. The use of the reagent combination as described in any one of claims 1 to 3 and / or the culture medium as described in any one of claims 4 to 6 in the preparation of neural mesodermal progenitor cells.

8. The use of the reagent combination as described in any one of claims 1 to 3 and / or the culture medium as described in any one of claims 4 to 6 in the preparation of neuromuscular organoids.

9. A method for preparing neuromuscular organoids, characterized in that, Includes the following steps: S1: After seeding induced pluripotent stem cells, induce differentiation culture for 1 day, culture with double inhibition for 1.5 days, and then transfer to a new culture vector; S2: Culture the cells that have completed differentiation in S1, passage them, expand the culture, and obtain the neuromuscular organoids; The induction described in S1 uses the basal medium, CHIR99021, and bFGF as described in claim 5 or 6; The dual inhibition described in S1 employs the basal culture medium, CHIR99021, bFGF, SB431542, and LDN193189 as described in claim 5 or 6.

10. The preparation method according to claim 9, characterized in that, The concentration of CHIR99021 in the induction is 5 µM; the concentration of CHIR99021 in the dual inhibition is 10 µM.

11. The preparation method according to claim 9 or 10, characterized in that, The new culture medium described in S1 includes the Aggrewell system.

12. Neuromuscular organoids obtained by the preparation method according to any one of claims 9 to 11.

13. The use of the neuromuscular organoid as described in claim 12 in any of the following: (a) Constructing an organoid model of amyotrophic lateral sclerosis (ALS); (b) Constructing drug models for screening and / or evaluating amyotrophic lateral sclerosis (ALS); (c) Screening for drugs for amyotrophic lateral sclerosis (ALS); (d) Treatment of amyotrophic lateral sclerosis (ALS); (e) Prepare products for the treatment of amyotrophic lateral sclerosis (ALS).

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