Method for constructing mouse embryo-derived hypothalamic organ model and application of mouse embryo-derived hypothalamic organ model

Abstract

The invention belongs to the technical field of biomedicine, and relates to a method for constructing a mouse embryo-derived hypothalamic organ model and application, the invention proposes that a mouse embryo is used for constructing a hypothalamic organ for the first time, and the mouse embryo has a definite development time sequence and genetic operability; the naturally retained vascular endothelial cells and multilineage precursor cells provide unique advantages for constructing a three-dimensional organ containing multiple nucleuses such as a preoptic region, a paraventricular nucleus, an arcuate nucleus and a supraoptic nucleus, and the model not only breaks through the nucleus coverage limitation of the existing iPSCs organ, but also can be used for constructing the three-dimensional organ containing multiple nucleuses such as the preoptic region, the paraventricular nucleus, the arcuate nucleus, the supraoptic nucleus and the like by reconstructing a nerve-blood vessel unit. High-fidelity simulation of complex physiological functions of the hypothalamus is realized, and an irreplaceable technical platform is provided for mechanism analysis of neuroendocrine diseases and high-throughput screening of drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a method for constructing a mouse embryo-derived hypothalamic organoid model and its application. Background Art

[0002] As the neuroendocrine hub for the body's homeostatic regulation, the hypothalamus regulates physiological processes such as energy metabolism, body temperature balance, reproductive cycle, and circadian rhythm by integrating neural and endocrine signaling networks. The complexity of its functions stems from its highly specialized cell composition, including key functional nuclei such as the preoptic area, paraventricular nucleus, arcuate nucleus, and supraoptic nucleus. The neuroendocrine neurons in these nuclei have the dual functional characteristics of neural signal transmission and hormone secretion, and together with glial cells, vascular endothelial cells, etc., they form sophisticated functional units. In order to deeply analyze how such units work together to achieve homeostatic regulation, traditional research methods face fundamental bottlenecks: although animal models can reflect overall physiological functions, the experimental cycle is long and it is difficult to achieve dynamic mechanism tracking with cellular resolution. The two-dimensional cell culture system cannot simulate its three-dimensional microenvironment and multi-nuclear group interactions, which greatly limits the study of the mechanism of its core function.

[0003] Although existing arcuate nucleus organoids constructed from human induced pluripotent stem cells (iPSCs) offer a new avenue for hypothalamic research, they can only simulate the functional properties of a single nucleus (the arcuate nucleus) and cannot recapitulate the coordinated neuroendocrine network of multiple nuclei, including the preoptic area, paraventricular nucleus, and supraoptic nucleus. Furthermore, these organoids lack key microenvironmental components such as vascular endothelial cells and microglia, resulting in structural and functional integrity far inferior to that of real tissue in vivo. Therefore, the development of innovative models that can fully simulate the function of multiple hypothalamic nuclei and the neurovascular microenvironment is an urgent need to overcome current research bottlenecks. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a method and application for constructing a mouse embryonic hypothalamic organoid model. This method directly utilizes mouse embryonic hypothalamic tissue to induce its self-organization and development in an in vitro three-dimensional (3D) culture system to form hypothalamic organoids with complex spatial structure and physiological functions. This method overcomes the problems of existing hypothalamic organoid models (such as human pluripotent stem cell-derived models), such as long construction cycle, unstable efficiency, and difficulty in accurately simulating the hypothalamic developmental microenvironment and function in mice.

[0005] The technical solution provided by the present invention is as follows:

[0006] The present invention provides a method for constructing a mouse embryonic hypothalamic organoid model, comprising the following steps: (1) Obtain hypothalamic tissue from mouse embryos at gestational day 17 to 19; (2) Placing the obtained hypothalamic tissue into organoid culture medium for three-dimensional dynamic culture; The procedures for obtaining hypothalamic tissue include: S1. Select FVB / NJ strain pregnant mice at 17-19 days of gestation and euthanize them. S2. Dissect the uterus under sterile conditions and collect embryos using Hibernate-E medium containing 2% v / v B-27 and GlutaMAX. S3. Locate the embryonic mandible under a dissecting microscope, cut open to expose the ventral side of the brain, and precisely separate the hypothalamic tissue. S4. Rinse the isolated hypothalamic tissue with Hibernate-E medium containing supplements and cut it into tissue blocks with a diameter of 1 mm.

[0007] Furthermore, the precise separation of the hypothalamic tissue in step S3 includes: excluding the anterior thalamic tissue, retaining only the hypothalamic region located behind the medial ganglionic eminence and in front of the midbrain and sensory thalamus, and performing fine dissection using ophthalmic forceps and microscissors.

[0008] Furthermore, the organoid culture medium is prepared based on Advanced DMEM / F12 and Neurobasal basal medium mixed in a 1:1 volume ratio, and the following cytokines are added: 10 U / mL penicillin-streptomycin, 1× GlutaMax, 1× B27 without vitamin A, 1× N2 supplement and 1× MEM non-essential amino acid solution.

[0009] Furthermore, the three-dimensional dynamic culture operation in step (2) includes: The obtained hypothalamic tissue pieces were resuspended in organoid culture medium and seeded into 6-well plates; The cells were placed in a 37°C, 5% CO2 incubator and cultured using an orbital shaker with continuous rotation at 100 rpm.

[0010] Furthermore, step (2) also includes an organoid culture medium replacement step: fresh organoid culture medium is replaced every 2 days, and lysed single cells are removed simultaneously during the replacement to retain the spherical organoid structure.

[0011] Furthermore, step (2) also includes an organoid formation verification step: after 2 weeks of culture, spherical organoid structures with clear boundaries are observed under a microscope.

[0012] The present invention also provides the use of hypothalamic organoids constructed by the above method in neural development research.

[0013] The present invention also provides the application of hypothalamic organoids constructed by the above method in the study of metabolic regulation mechanisms.

[0014] The present invention also provides the use of hypothalamic organoids constructed by the above method in establishing a neuroendocrine disease model.

[0015] The present invention also provides the use of hypothalamic organoids constructed by the above method in establishing a drug screening platform.

[0016] Beneficial effects:

[0017] This invention proposes for the first time a new strategy for constructing hypothalamic organoids using mouse embryos. Mouse embryos have a clear developmental timing and genetic operability, and their naturally retained vascular endothelial cells and multi-lineage precursor cells provide unique advantages for constructing three-dimensional organoids containing multiple nuclei such as the preoptic area, paraventricular nucleus, arcuate nucleus, and supraoptic nucleus. This model not only breaks through the limitations of nuclear coverage of existing iPSCs organoids, but also achieves high-fidelity simulation of the complex physiological functions of the hypothalamus by reconstructing the neurovascular unit, providing an irreplaceable technical platform for the analysis of the mechanisms of neuroendocrine diseases and high-throughput drug screening. In the future, by establishing standards for the identification of nuclear markers to promote technical standardization, this platform will eventually be transformed into a high-fidelity physiological simulation system for the research and drug development of diseases such as obesity and hypertension.

[0018] This invention successfully established for the first time a hypothalamic organoid model directly derived from mouse embryos. This model fills the gap in in vitro research models of the mouse hypothalamus and provides a powerful new in vitro research platform for in-depth exploration of the developmental mechanism, physiological function, disease model construction and drug screening of the mouse hypothalamus.

[0019] The model of the present invention can self-organize and maintain the key spatial partitioning structures of the hypothalamus (such as the arcuate nucleus, paraventricular nucleus, supraoptic nucleus and other areas) and cell composition diversity (including neurons, glial cells, etc.) in in vitro culture, highly simulating the complex microenvironment of the hypothalamus in vivo.

[0020] This study used calcium imaging to demonstrate that neurons within the organoids exhibited spontaneous and stimulus-induced calcium signaling activity, demonstrating the electrophysiological activity of their neural networks. More importantly, by stimulating the organoids with leptin, a key hormone regulating metabolism, the researchers observed changes in the activity of specific nuclei within the organoids (such as ARC neurons) and neuropeptide expression, demonstrating that the model recapitulates key neuroendocrine regulatory functions of the hypothalamus (such as energy metabolism regulation) in vitro and demonstrates genuine physiological relevance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is a flow chart of the culture of mouse hypothalamic organoids and morphological diagrams at key time points during the culture process. A is a schematic diagram of the dissection of mouse embryo isolation, and BG are organoid morphology images of mouse hypothalamic organoids under an optical microscope on days 0, 1, 2, 4, 8, 15, and 26 of primary culture. When photographing, the organoids were in matrigel. Scale bar: 100 μm.

[0022] Figure 2 This is the real-time fluorescence quantitative PCR result of hypothalamic organoids on the 14th day of primary culture, where A is the cultured hypothalamic organoids Pomc Figure 2. mRNA expression detection results. Figure B shows cultured hypothalamic organoids. Npy Figure 3. mRNA expression detection results. Figure C shows cultured hypothalamic organoids. Mch Figure 2 shows the results of mRNA expression detection. D shows the cultured hypothalamic organoids. Nkx2.1 Figure 2. mRNA expression detection results. E is the cultured hypothalamic organoids. Six3 Figure 2. mRNA expression detection results. Figure F shows cultured hypothalamic organoids. Rax Figure 2. mRNA expression detection results.

[0023] Figure 3 This is the immunofluorescence result of hypothalamic organoids on the 14th day of primary culture, where A is the cultured hypothalamic organoids PAX6 Immunofluorescence results, B is the cultured hypothalamic organoids NEUN Immunofluorescence results, C is the cultured hypothalamic organoids GAD65 Immunofluorescence results, D is the cultured hypothalamic organoids BDNF Immunofluorescence results, scale bar: 20 μm.

[0024] Figure 4 The results of calcium signal detection in hypothalamic organoids on the 14th day of primary culture are shown. A is a bright-field photograph of the organoid, B is a fluorescence photograph of the calcium signal before adding KCl, and C is a fluorescence photograph of the calcium signal after adding KCl. The scale bar is 2 mm. D is a statistical graph of the calcium signal.

[0025] Figure 5 The expression changes of downstream JAK2 / STAT3 signaling pathway proteins were detected in hypothalamic organoids on the 14th day of primary culture under leptin stimulation. A is the JAK2 / STAT3 protein expression detection band of organoids under physiological saline and leptin stimulation, respectively, and B is the protein quantitative analysis of Figure A. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0027] Example 1:

[0028] 1. Mouse Embryo Isolation Experimental Method

[0029] 1.1 Animal mating and gestation period determination

[0030] Eight-week-old FVB / NJ mice were selected and co-bred at a sex ratio of 1:1 (or 2:1) between 6:00 PM and 8:00 PM daily. A vaginal plug was examined at 8:00 AM the following day, and the day of its discovery was designated embryonic day 0.5 (E0.5). Observation was continued until E18.5 for subsequent experiments.

[0031] 1.2 Embryo Collection and Processing

[0032] Tissue isolation and dissection

[0033] 1) Experimental preparation:

[0034] Select healthy pregnant mice at gestational age of 18.5 days (E18.5) to ensure normal embryonic development.

[0035] Prepare the following reagents and tools:

[0036] Hibernate-E medium (Thermo Fisher Scientific) was supplemented with 2% v / v B-27 supplement and GlutaMAX supplement.

[0037] 10 cm cell culture dish, ophthalmic scissors, ophthalmic forceps, dissecting needles and other fine surgical instruments, sterile operating table, microscope, incubator (37°C, 5% CO2).

[0038] 2) Embryo collection:

[0039] E18.5 mouse embryos were euthanized under sterile conditions in accordance with relevant ethical regulations.

[0040] Embryos were collected using Hibernate-E medium (ThermoFisher Scientific) supplemented with 2% v / v B-27 supplement and GlutaMAX supplement and placed in sterile culture dishes to ensure embryonic integrity and a sterile environment.

[0041] 3) Dissection operation:

[0042] Place the embryo under a dissecting microscope and gently secure it using ophthalmic forceps.

[0043] Make a small incision below the embryo's jaw to expose the ventral portion of the brain.

[0044] Tissue located posterior to the medial ganglionic eminence and anterior to the midbrain and sensory thalamus was dissected out.

[0045] Precisely exclude the anterior thalamic tissue and collect only the hypothalamic tissue to minimize damage to the hypothalamic tissue during dissection.

[0046] The collected embryonic hypothalamic tissue was transferred to a 10-cm sterile culture dish, and an appropriate amount of Hibernate-E medium containing 2% v / v B-27 supplement and GlutaMAX supplement was added, and the dish was gently rinsed to remove residual blood and tissue debris.

[0047] 4) Tissue cutting:

[0048] Place the culture dish containing hypothalamic tissue under a dissecting microscope.

[0049] Use ophthalmic scissors to cut the hypothalamic tissue into small pieces, each approximately 1 mm in diameter. Be gentle during cutting to avoid excessive damage to the tissue.

[0050] Transfer the cut tissue pieces to a new culture dish for subsequent culture.

[0051] 2. Generation and Culture of Hypothalamic Organoids

[0052] 2.1 Resuspending tissue chunks:

[0053] Gently transfer the dissected hypothalamic tissue blocks into a centrifuge tube.

[0054] Add to optimized organoid culture medium, which consists of a 1:1 volume ratio of Advanced DMEM / F12 and Neurobasal medium, supplemented with the following components:

[0055] 10 U / mL penicillin-streptomycin (to ensure a sterile environment and prevent microbial contamination);

[0056] 1× GlutaMax (provides essential glutamine to support cell metabolism);

[0057] 1 x Vitamin A-free B27 supplement (provides nutrients necessary for cell growth and development, but avoids the potential effects of Vitamin A on neural differentiation);

[0058] 1× N2 supplement (to further support the growth and differentiation of nerve cells);

[0059] 1×MEM non-essential amino acid solution (promotes cell metabolism and protein synthesis);

[0060] Gently vortex or pipette to mix to resuspend tissue chunks.

[0061] 2.2 Inoculation and cultivation:

[0062] The mixture of resuspended hypothalamic tissue pieces and culture medium was seeded into a 6-well plate, and an appropriate amount of the mixture was added to each well to ensure that the tissue pieces were evenly distributed.

[0063] Place the 6-well plate in a 37°C, 5% CO2 incubator and culture with continuous rotation at 100 rpm on an orbital shaker. This dynamic culture method promotes uniform stress distribution across the tissue and adequate nutrient exchange, which is beneficial for the formation and development of organoids.

[0064] 2.3 Culture medium replacement:

[0065] Change the organoid culture medium every two days. When changing the organoid culture medium, please note the following points:

[0066] Gently aspirate old culture medium to avoid damaging tissue fragments and formed organoids.

[0067] Add fresh pre-warmed culture medium to ensure that the cells are in a suitable growth environment.

[0068] Tissue that has not formed organoids will be lysed into single cells, which can be discarded by changing the medium, thereby enriching tissue with organoid structure.

[0069] 2.4 Observation of organoid formation:

[0070] Two weeks after inoculation of the tissue blocks, the formation of spherical organoid-like structures with clear boundaries was observed under a microscope, providing a suitable model for subsequent experimental studies.

[0071] Example 2:

[0072] 1. Immunohistochemistry and staining

[0073] 1.1 Sample washing and fixation:

[0074] Transfer the cultured or processed hypothalamic organoid samples to a centrifuge tube.

[0075] Wash the sample thoroughly three times with phosphate-buffered saline (1× PBS, pH = 7.4). For each wash, add sufficient PBS to gently resuspend the sample, incubate at room temperature (approximately 20–25°C) for 2–5 minutes, then centrifuge at 1000 g for 5 minutes and carefully discard the supernatant.

[0076] After washing, add pre-cooled 4% w / v paraformaldehyde (dissolved in PBS, pH = 7.4) fixative to the sample, ensuring that the sample is completely immersed.

[0077] Place the sample in a 4°C refrigerator for fixation and incubate for 16 to 24 hours (overnight).

[0078] 1.2 Dehydration:

[0079] After fixation, discard the fixative.

[0080] Wash the sample once with PBS (same method as in step 1) to remove residual fixative.

[0081] The samples were placed in different concentration gradient ethanol aqueous solutions for dehydration treatment: 70% v / v ethanol, soaking for 15-30 min; 85% v / v ethanol, soaking for 15-30 min; 95% v / v ethanol, soaking for 15-30 min; 100% v / v anhydrous ethanol, soaking twice, each time for 15-30 min.

[0082] All dehydration steps were performed at room temperature (approximately 20-25°C).

[0083] 1.3 Transparency:

[0084] After dehydration is complete, completely discard the ethanol.

[0085] Transfer the sample to xylene for clearing: Soak the sample in xylene twice, each time for 15-30 minutes (at room temperature), until the sample is completely transparent, ensuring that the ethanol is completely replaced.

[0086] 1.4 Paraffin embedding and sectioning:

[0087] After clearing, the xylene was aspirated and discarded.

[0088] Place the sample in molten paraffin (melting point range 56-58°C) for wax immersion. Usually, fresh paraffin needs to be replaced 2-3 times, with each immersion lasting 30-60 minutes (at a temperature slightly above the paraffin melting point, approximately 60°C).

[0089] Place the fully wax-soaked sample in an embedding mold, add fresh molten paraffin, and cool and solidify to form a paraffin block.

[0090] The embedded tissue blocks were serially sectioned using a paraffin microtome with a section thickness of 5 μm.

[0091] Float the cut paraffin sections in warm water (about 40~45℃) and flatten them.

[0092] Use a glass slide treated with poly-lysine or 3-aminopropyltriethoxysilane (APES) to pick up the flattened sections.

[0093] The slides with sections attached were placed in a 37°C oven to dry overnight to allow the sections to adhere firmly.

[0094] 1.5 Immunofluorescence staining:

[0095] 1.5.1 Dewaxing and hydration:

[0096] Immerse the dried sections in the following solutions in sequence: xylene I: 10 min; xylene II: 10 min; 100% v / v ethanol I: 5 min; 100% v / v ethanol II: 5 min; 95% v / v ethanol: 5 min; 85% v / v ethanol: 5 min; 70% v / v ethanol: 5 min; and deionized water: 5 min. All steps should be performed at room temperature (approximately 20–25°C).

[0097] 1.5.2 Permeabilization and blocking:

[0098] Absorb the water and draw a hydrophobic circle around the tissue section with a PAP pen.

[0099] Add a sufficient amount of blocking solution to the tissue section. The blocking solution contains: 1× PBS, 5% w / v bovine serum albumin (BSA), and 0.3% v / v Triton X-100 (for permeabilization of cell membranes).

[0100] Place the slides in a humidified chamber and incubate at room temperature (approximately 20-25°C) for 2 hours.

[0101] 1.5.3 Primary Antibody Incubation:

[0102] After blocking, discard the blocking solution (no need to wash).

[0103] Apply the primary antibody working solution diluted in PBS containing 2% w / v BSA and 0.1% v / v Tween-20 (PBST) to the tissue section. The primary antibody dilution ratio is preferably 1:200.

[0104] Place the slides in a humidified chamber and incubate in a 4°C refrigerator overnight (16-24 hours).

[0105] 1.5.4 Washing:

[0106] After the primary antibody incubation is completed, discard the primary antibody solution.

[0107] Wash the slides three times with PBST (PBS containing 0.1% v / v Tween-20), soaking for 5-10 minutes each time, with gentle shaking on a shaker at room temperature.

[0108] 1.5.5 Secondary Antibody Incubation:

[0109] Discard the last wash solution.

[0110] Add fluorescently labeled secondary antibody working solution diluted in PBST containing 2% w / v BSA to the tissue section. The preferred dilution ratio of the secondary antibody is 1:1000.

[0111] Place the slides in a humidified chamber and incubate at room temperature (approximately 20-25°C) in the dark for 1 hour.

[0112] 1.5.6 Washing and sealing:

[0113] After the secondary antibody incubation is completed, discard the secondary antibody solution.

[0114] Wash the slides three times with PBST, each time for 5-10 min (same as step 1.5.4).

[0115] Optional: If nuclear staining is desired, add DAPI (1 μg / mL in PBS or PBST) and incubate for 5–10 min. Wash the cells three times with PBS or PBST, each for 5 min.

[0116] Discard most of the liquid and carefully blot the liquid around the tissue with filter paper (avoid touching the tissue area).

[0117] Add an appropriate amount of anti-fluorescence quenching mounting medium (for example, ProLong Gold, Vectashield, etc. with or without DAPI) on the tissue.

[0118] Carefully cover with a coverslip to avoid creating air bubbles. Allow the mounting medium to solidify at room temperature in the dark, or as directed in the manufacturer's instructions.

[0119] 1.6 Image Acquisition:

[0120] After the mounting medium was completely solidified / dried, images of the stained sections were acquired using a confocal laser scanning microscope.

[0121] Select appropriate laser and filter combinations based on the excitation / emission spectra of the fluorescent secondary antibodies and nuclear stains used.

[0122] Use a high numerical aperture (NA) objective (e.g., a 63x oil objective) for high-resolution imaging.

[0123] Set the appropriate Z-stack layer number and step distance (e.g., 0.5–1 μm) as needed to obtain three-dimensional information.

[0124] Adjust laser intensity, gain, offset and other parameters to obtain the best signal-to-noise ratio and avoid signal overexposure or quenching.

[0125] Save the original image data for subsequent analysis.

[0126] 2. Calcium Ion Imaging Experiment

[0127] Transfer the organoids to a 6-cm dish containing fresh organoid culture medium. Add the Fluo-8 calcium fluorescent probe and incubate the organoids at 37°C for 30 minutes, then wash three times with PBS. Transfer the organoids to a new dish. Record calcium activity using confocal microscopy. Stimulate neural activity by adding 40 mM KCl solution, and record calcium activity again 1 minute later.

[0128] 2.1 Organoid Processing

[0129] Gently transfer the cultured hypothalamic organoids from a regular culture dish to a sterile 6 cm dish containing fresh organoid culture medium. Ensure that the organoids are evenly distributed in the culture medium and avoid mechanical damage during transfer.

[0130] After transferring to a new culture dish, allow the organoids to acclimate to the new environment in a 37°C, 5% CO2 incubator for approximately 15 minutes to ensure their physiological state is stable.

[0131] 2.2 Calcium fluorescent probe staining:

[0132] Prepare Fluo-8 calcium fluorescent probe working solution by diluting and preparing according to the reagent instructions. Fluo-8 is a highly sensitive calcium ion fluorescent indicator that specifically binds to intracellular calcium ions and emits fluorescence.

[0133] Add an appropriate amount of Fluo-8 calcium fluorescent probe working solution to the culture dish, ensuring that the organoids are completely immersed in the culture medium.

[0134] Incubate the culture dish in a 37°C incubator for 30 minutes. Ensure the dish remains level during incubation to prevent uneven dye distribution. Fluo-8 incubation at 37°C allows it to effectively penetrate into organoid cells, bind to intracellular calcium ions, and provide a fluorescent signal for subsequent calcium imaging.

[0135] 2.3 Washing steps:

[0136] After incubation, gently wash the organoids three times with pre-warmed PBS (phosphate-buffered saline). For each wash, gently aspirate the supernatant, add PBS, and gently rock the dish on a shaker at low speed for approximately 1 minute to remove any unbound fluorescent probe and any background fluorescence. This wash step is crucial for reducing background fluorescence and improving the signal-to-noise ratio in subsequent imaging.

[0137] 2.4 Basal calcium activity recording

[0138] Transfer the washed organoids to a new 6 cm sterile culture dish and add an appropriate amount of fresh organoid culture medium to ensure that the organoids maintain a suitable growth environment during imaging.

[0139] Place the dish containing the organoids on the stage of a confocal microscope. Adjust the microscope parameters to obtain the best fluorescence signal.

[0140] Before adding the stimulant, first record the basal calcium activity of the organoid. Scan the organoid continuously for approximately 2 minutes to observe and record the spontaneous activity of intracellular calcium ions. This step provides the organoid's resting calcium level, providing a reference for subsequent calcium changes after stimulation.

[0141] 2.5 KCl stimulation and calcium ion activity recording:

[0142] A 40 mM KCl (potassium chloride) solution was prepared as a stimulant for neural activity. KCl increases the extracellular potassium ion concentration, causing cell membrane depolarization, thereby activating voltage-gated calcium channels and increasing the intracellular calcium ion concentration.

[0143] Carefully add 40 mM KCl solution to the culture dish, ensuring that the organoids are completely submerged in the KCl-containing medium.

[0144] One minute after the addition of KCl solution, organoid calcium activity was again recorded using confocal microscopy for approximately 3 minutes.

[0145] 2.6 Data processing and analysis:

[0146] After the experiment, the collected images and data were imported into professional image analysis software to quantitatively analyze the fluorescence signal, calculate the changes in intracellular calcium ion concentration, and compare the differences before and after KCl stimulation.

[0147] 3. Real-time quantitative PCR (qRT-PCR)

[0148] 3.1 RNA extraction from hypothalamic organoids

[0149] 3.1.1 Sample preprocessing

[0150] Gently wash hypothalamic organoids three times with ice-cold 1× PBS to remove residual culture medium.

[0151] Transfer the organoids to a 2 mL grinding tube and centrifuge at 2000 rpm for 5 min to collect the precipitate.

[0152] 3.1.2 RNA lysis and isolation

[0153] Add 1 mL of Trizol lysis buffer, mix thoroughly by pipetting, and place on ice for 30 min to ensure complete lysis.

[0154] Add 200 μL of chloroform, shake vigorously for 15 seconds to mix, and centrifuge at 4°C and 13,000 rpm for 15 minutes to allow the RNA to enter the upper aqueous phase.

[0155] Carefully pipette 450 μL of supernatant into a 1.5 mL RNase-free EP tube, add an equal volume of isopropanol, mix gently, and centrifuge at 13,000 rpm at 4°C for 15 min to precipitate RNA.

[0156] 3.1.3 RNA purification

[0157] The supernatant was discarded, and 1 mL of 75% v / v ice-cold ethanol was added to wash the precipitate. The precipitate was centrifuged at 13,000 rpm at 4°C for 10 min.

[0158] Discard the ethanol, place the EP tube upside down on sterile filter paper, and dry it at room temperature for 5-10 min until the RNA precipitate becomes transparent.

[0159] Add appropriate amount of DEPC water to dissolve RNA precipitate, vortex and incubate at 37°C for 30 min to promote dissolution.

[0160] 3.1.4 RNA quantification and reverse transcription

[0161] RNA concentration and purity were determined using Nanodrop (A260 / A280 ratio should be between 1.8 and 2.0).

[0162] Use a two-step reverse transcription kit (such as Takara PrimeScript RT Reagent Kit) according to the instructions:

[0163] Step 1: 1 μg RNA was treated with gDNA Eraser to remove genomic DNA contamination.

[0164] Step 2: Reverse transcription to synthesize cDNA. The reaction conditions are 37℃ for 15 min and 85℃ for 5 sec.

[0165] The cDNA product was temporarily stored at 4°C for later use. For long-term storage, it is recommended to store it at -20°C.

[0166] 3.2 qRT-PCR detection

[0167] 3.2.1 Template preparation

[0168] The cDNA was diluted 25-fold with nuclease-free ddH2O and used as a qPCR template.

[0169] 3.2.2 Reaction system preparation

[0170] Use SYBR Green Master Mix (such as Novozymes AceQ qPCR SYBR Green Master Mix) to prepare 10 μL of the following system (set up 3 technical replicates for each sample), see Table 1:.

[0171] Table 1 qRT-PCR reaction system

[0172]

[0173] 3.2.3 qRT-PCR Program Settings

[0174] The following program was run using the Roche LightCycler 480II instrument:

[0175] Initial denaturation: 95°C for 30 seconds;

[0176] Amplification cycle (40 cycles): 95°C 10 sec → 60°C 30 sec;

[0177] Melting curve analysis: 95°C 15 sec → 60°C 60 sec → 95°C 15 sec (continuously collect fluorescence signals).

[0178] The primer sequences are shown in Table 2:

[0179] Table 2 Summary of primer sequence information

[0180]

[0181] 3.2.4 Data Analysis

[0182] by Gapdh As an internal reference gene, 2 -ΔΔCt The relative expression level of the target gene was calculated by the method.

[0183] 4. Detection of JAK2 / STAT3 protein expression in hypothalamic organoids

[0184] 4.1 Protein extraction

[0185] Preparation of reagents and consumables: RIPA (1 M Tris-HCl pH = 8.0, 5 M NaCl, 10% v / v SDS, 10% v / v NP40, 0.5% v / v sodium deoxycholate, 2 M MgCl); protease / phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, 25 mM sodium β-glycerophosphate, 50 μg / mL PMSF, protease inhibitors); 5× protein loading buffer; 1.5 mL eppendorf tubes; 200 μL eppendorf tubes; BCA protein concentration assay kit (A:23228, B:1859078, Thermo Fisher Scientific); 96-well plate.

[0186] Gently transfer the cultured hypothalamic organoids from a regular culture dish to a sterile 6 cm dish containing fresh organoid culture medium. Ensure that the organoids are evenly distributed in the culture medium and avoid mechanical damage during transfer.

[0187] After transferring to a new dish, allow the organoids to acclimate to the new environment in a 37°C, 5% CO2 incubator for approximately 15 minutes to ensure their physiological state is stable.

[0188] Leptin stimulation:

[0189] Prepare the leptin working solution by diluting and preparing according to the reagent instructions. Leptin is a protein hormone primarily secreted by adipocytes. Its primary target is the hypothalamus in the brain. Leptin activates the constitutively bound JAK2 kinase via its receptor, which then phosphorylates the receptor and the STAT3 transcription factor. This ultimately drives STAT3-dependent gene expression, enabling its physiological regulation of energy balance and metabolism.

[0190] Add 2 μg / mL leptin working solution to the culture dish, ensuring that the organoids are completely immersed in the culture medium.

[0191] Incubate the culture dish in a 37°C incubator for 60 minutes. Ensure the dish remains level during incubation to prevent uneven leptin distribution. Leptin incubation at 37°C allows it to effectively bind to the receptor and activate the downstream signaling pathway, JAK2 / STAT3.

[0192] For each wash, gently aspirate the supernatant, add PBS, and gently rock the dish on a shaker at low speed for approximately 1 minute to ensure removal of unbound fluorescent probe and any background fluorescence. This wash step is crucial for reducing background fluorescence and improving the signal-to-noise ratio in subsequent imaging.

[0193] Gently remove the supernatant, add PBS, wash three times, add 100 mL of protein lysis buffer, transfer to a grinding tube, and place on ice for full lysis for 30 min.

[0194] Centrifuge at 14,000 rpm at 4°C for 15 min.

[0195] The supernatant was transferred to a new 1.5 ep tube, and 5 μL of sample was diluted 10-fold for protein concentration detection. The whole operation was carried out on ice.

[0196] Take a 96-well plate, set up 2 blank control wells, 7 standard test wells, and the rest are sample test wells.

[0197] Sequentially add 40 μL of ddH2O to the blank well and the standard test well, and add 45 μL of ddH2O to the sample test well.

[0198] Add 10 μL of ddH2O to the blank wells, add 10 μL of 2 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL protein standards to the standard wells, add 5 μL of the test protein to the protein test wells, and fill up to 50 μL with ddH2O.

[0199] Prepare the detection reaction solution according to the kit operation. Add solution A and solution B in the detection reaction solution at a ratio of 1:50 and mix well.

[0200] Add 200 μL of reaction solution to each well and incubate at 37°C for 30 min.

[0201] Read the OD value of each test well at 562 nm using a full-function multi-band microplate reader. Plot a regression curve based on the standard curve, comparing the OD value of each test well with its corresponding protein concentration. Calculate the protein concentration based on the regression equation and the OD value of the test well.

[0202] The concentration of each sample was calibrated to the same level using ddH2O according to the protein concentration.

[0203] Take 100 μL of the calibrated protein solution, add 25 μL of protein loading buffer, and boil the sample in a 95°C water bath for 10 min. After the sample cools naturally, vortex and mix thoroughly, then aliquot and store in a -80°C refrigerator.

[0204] 4.2 Western Blot

[0205] (1) Preparation of reagents and consumables: 1× electrophoresis buffer (weigh 3.03 g Tris-base, 18.77 g Gly, 1.0 g SDS, dissolved in 1 L ddH2O); 1× transfer buffer (weigh 5.80 g Tris-base, 2.90 g Gly, 0.37 g SDS, 200 mL methanol, dissolved in 1 L ddH2O); 10% separation gel (2.4 mL ddH2O, 1.25 mL 1.5 M Tris-HCl pH = 8.8, 1.25 mL 40% v / v acrylamide, 2 μL TMED, 50 μL 10% v / v SDS, 50 μL ammonium persulfate); 4% stacking gel (1.21 mL ddH2O, 500 μL 1.0 M Tris-HCl pH = 6.8, 250 μL 40% v / v acrylamide, 2 μL TMED, 20 μL 10% v / v SDS, 20 μL ammonium persulfate); methanol; 1× TBST (15 mL 5 M NaCl, 10 mL 1.5 M Tris-HCl (pH 7.5), 2.5 mL 20% v / v Tween 20 dissolved in 500 mL ddH2O); developer (34094, 34080, Thermo Fisher Scientific); skim milk powder; PVDF membrane; glass plate for gel casting; comb; electrophoresis apparatus; membrane transfer apparatus; filter paper; sponge; tweezers; glass rod; membrane washing box.

[0206] (2) Wash a 1.0 mm thick glass plate with tap water, let it dry, and then mount it on a gel rack. Add the ingredients in the recipe to prepare the separation gel mixture and mix thoroughly. Each gel needs to be filled with approximately 5.0 mL of the mixture. Add ddH2O to the filled separation gel and press it into place. Slightly shake the glass plate left and right until the separation gel and ddH2O are flush with the dividing line.

[0207] (3) After the separation gel is solidified, prepare the concentrated gel according to the proportion of ingredients and pour it into the upper end of the separation gel. Each concentrated gel needs to be filled with about 2.0 mL of the mixture. After filling the concentrated gel, insert a clean and dried comb.

[0208] (4) Connect the electrophoresis device, install the prepared polyacrylamide gel into the electrophoresis device, and pull out the comb vertically. Add 500 mL of the newly prepared 1× electrophoresis buffer to the sample well and use a pipette to remove the bubbles at the top of the sample well.

[0209] (5) Determine the loading volume based on the target protein's expression abundance in the tissue. Add 3 μL of protein marker to both ends of the loading wells, and add 1× protein loading buffer to the blank wells without sample. Start the electrophoresis power supply and operate at a constant voltage of 60 V for 30 min. When the protein sample runs to the boundary between the separation gel and the stacking gel and forms a straight line, switch the voltage to 120 V and continue operating. Turn off the power supply when the bromophenol blue in the sample runs to the bottom of the separation gel.

[0210] (6) Cut a PVDF membrane of approximately 7.5 cm x 9 cm as required and activate it in methanol for 5 min. Remove the run-through protein gel by wet prying and place it on the transfer device with a sandwich structure of blackboard-sponge-filter paper-gel-membrane-filter paper-sponge-red board. Pour the prepared transfer solution containing methanol into the transfer device and transfer the entire transfer device to a foam box filled with crushed ice. Turn on the power supply and set the constant voltage of 100 V for 90 min. The specific transfer time should be determined according to the specific protein.

[0211] (7) The PVDF membrane that has completed the transfer is taken out of the transfer device, and the band near the target protein is cut according to the mark. It is transferred to 5% w / v skim milk powder solution and blocked at room temperature for 1 h.

[0212] (8) 4 mL of primary antibody (pJAK2 / JAK2 / pSTAT3 / STAT3 / Actin) diluted with 5% w / v skim milk powder was added to the antibody incubation box. The protein bands were placed in the incubation box and incubated on a shaker at 4 °C overnight.

[0213] (9) Rinse the strips three times in 1× TBST on a shaker at room temperature, 10 min each time.

[0214] (10) Take 4 mL of the secondary antibody (goat anti-rabbit IgG-HRP / goat anti-mouse IgG-HRP) diluted with 5% w / v skim milk powder and put it into the antibody incubation box. Place the protein band in the incubation box and incubate on a shaker at room temperature for 1 h.

[0215] (11) Repeat step 9.

[0216] (12) Select a suitable developer and prepare it immediately before use. Start the chemiluminescence image analyzer (Tanon-5200, Tianneng), open the gel electrophoresis image analysis software, and take photos.

[0217] (13) Use Image-J to semi-quantitatively analyze the grayscale values ​​of the target protein and the internal reference protein in each well. The grayscale value of the target protein in each well is calibrated with its own internal reference grayscale value to obtain the relative grayscale value of the target protein. Then, the relative grayscale value of the control group is corrected to 1 to calculate the relative expression of the target protein. The antibodies used are shown in Table 3:

[0218] Table 3 Summary of WB antibody information

[0219]

[0220] 5. Results and Discussion

[0221] The experimental results are as follows Figure 1 shown. Figure 1 Figure A in the middle shows the culture process of mouse hypothalamic organoids: mouse embryos at embryonic day 18.5 (E18.5) are taken, the hypothalamic tissue is separated by micromanipulation, and a single-cell suspension is prepared by mechanical dissociation. The cells are then inoculated into a special culture dish for three-dimensional suspension culture. Figure 1 The BG system recorded morphological changes at key time points during the culture process, including 0, 1, 2, 4, 8, 15, and 26 days. As can be seen from the figure, the organoids gradually began to assume a spherical shape after 8 days of culture, and by 15 and 26 days, they had formed more clearly defined spherical organoids, demonstrating that the cells in the culture system were able to self-assemble into compact organoids. Their morphological characteristics were as follows: 1) high-density, orderly cell arrangement; 2) an overall nearly spherical three-dimensional structure; and 3) typical tissue heterogeneity. Together, these observations confirm that the culture method described herein can effectively support the in vitro construction and maintenance of mouse hypothalamic organoids.

[0222] The experimental results are as follows Figure 2 As shown. To verify the hypothalamic specificity of the constructed organoids, we evaluated their functional characteristics through a molecular marker analysis system. The experiment detected the following key indicators: (1) functional neuropeptides: including pro-opiomelanocortin (Pomc), neuropeptide Y (Npy) and melanin-concentrating hormone (Mch); (2) transcriptional regulatory factors: including NKX2.1, Six3 and Rax. Quantitative analysis showed that compared with natural hypothalamic tissue, the in vitro cultured organoids showed the following characteristic expression profiles: Pomc and Npy expression levels were significantly upregulated ( p < 0.05, p < 0.001), and Mch expression was significantly downregulated ( p < 0.05); Simultaneously, the expression levels of the transcription factors NKX2.1, Six3, and Rax all decreased to varying degrees. This molecular expression profile demonstrates that the organoid model constructed in this paper successfully retains key hypothalamic neuroendocrine functional properties, confirming that this in vitro culture system can effectively maintain the functional activity of hypothalamic organoids.

[0223] The experimental results are as follows Figure 3 To comprehensively evaluate the cellular composition and functional characteristics of hypothalamic organoids, we used immunofluorescence to analyze neural developmental markers. PAX6, mature neuron marker NEUN, γ-aminobutyric acid neuron marker GAD65 and neurotrophic factor BDNF were systematically tested. The results showed that: (1) PAX6 The results of the present study showed that the expression pattern of NEUN in organoids was regional, and its distribution characteristics were consistent with the expression patterns during embryonic hypothalamic development. (2) NEUN-positive cells were widely distributed in the organoids, confirming the presence of mature neuronal populations. (3) GAD65 expression detection confirmed the successful differentiation of inhibitory neurons. (4) The sustained expression of BDNF indicated that the organoids had the potential to maintain neural activity and synaptic plasticity. These immunofluorescence detection results were mutually confirmed with the aforementioned molecular marker analysis data, and jointly confirmed from the two levels of cell phenotype and function that the culture system described in the present invention can successfully construct an organoid model with typical hypothalamic tissue characteristics and functional activity.

[0224] The experimental results are as follows Figure 4 As shown. The hypothalamus contains neurons that can generate neural electrophysiological activities, so we tested whether neural electrophysiological activities can also be generated in organoids. In order to determine whether the organoids exhibit electrophysiological activity, we used a calcium imaging kit to detect the organoids. The kit uses Fluo-8 AM to detect the concentration of calcium ions in cells. The concentration of calcium ions is related to neural activity. By specifically binding to calcium ions with calcium ion fluorescent probes, the level of intracellular calcium ion concentration can be observed, and the situation of neural activity can be understood. Fluo-8 AM is an acetyl methyl ester derivative of Fluo-8, which has cell membrane permeability. After entering the cell, it reacts with free Ca 2+ Combined with the green fluorescence, the expression of green fluorescence can be observed using a fluorescence microscope. Using the kit, we can see that the organoids emit green fluorescence, indicating electrophysiological activity within the organoids. KCl can stimulate nerve cell excitation, so I added KCl to the organoid culture medium and observed an increase in fluorescence intensity. Statistically, we found that the fluorescence intensity of the hypothalamic organoids was greater after the addition of KCl than without it. This indicates that the hypothalamic organoids can generate neural electrophysiological activity and respond to external stimuli.

[0225] The experimental results are as follows Figure 5As shown. An important functional feature of the arcuate nucleus of the hypothalamus (ARC) is that its POMC neurons can respond to leptin stimulation and release α-melanocyte stimulating hormone (α-MSH). To verify the functional responsiveness of hypothalamic organoids cultured in vitro, we designed a leptin stimulation experiment: the experimental group was treated with 2 μg / mL leptin for 24 hours, and the control group was given an equal amount of normal saline. Western blot detection results showed that compared with the control group, the JAK2 phosphorylation level in the organoids of the leptin-treated group was significantly increased, and the STAT3 phosphorylation level was significantly enhanced. This result confirmed that: (1) the organoids constructed by this culture system completely retained the expression of leptin receptors; (2) the POMC neurons in the organoids had normal downstream signal transduction function; (3) the leptin-JAK2-STAT3 signaling pathway could be effectively activated in the organoids. This functional experiment, together with the above-mentioned morphological and molecular marker detection results, constitutes a complete chain of evidence, which fully proves that the hypothalamic organoids constructed by the present invention not only have tissue structural characteristics, but also have key physiological response functions, providing an ideal in vitro model for studying hypothalamic neuroendocrine regulation.

[0226] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for constructing a mouse embryonic hypothalamic organoid model, characterized in that: The following steps are involved: (1) Obtain hypothalamic tissue from mouse embryos at gestational day 17 to 19; (2) Placing the obtained hypothalamic tissue into organoid culture medium for three-dimensional dynamic culture; The procedures for obtaining hypothalamic tissue include: S1. Select FVB / NJ strain pregnant mice at 17-19 days of gestation and euthanize them. S2. Dissect the uterus under sterile conditions and collect embryos using Hibernate-E medium containing 2% v / v B-27 and GlutaMAX. S3. Locate the embryonic mandible under a dissecting microscope, cut open to expose the ventral side of the brain, and precisely separate the hypothalamic tissue. S4. Rinse the isolated hypothalamic tissue with Hibernate-E medium containing supplements and cut it into tissue blocks with a diameter of 1 mm. The precise separation of hypothalamic tissue in step S3 includes: excluding the anterior thalamic tissue and retaining only the hypothalamic region located behind the medial ganglionic eminence and in front of the midbrain and sensory thalamus, and performing fine dissection using ophthalmic forceps and microscissors; The organoid culture medium is prepared based on a 1:1 volume ratio of Advanced DMEM / F12 and Neurobasal basal medium, supplemented with the following cytokines: 10 U / mL penicillin-streptomycin, 1× GlutaMax, 1× B27 without vitamin A, 1× N2 supplement, and 1× MEM non-essential amino acid solution; The three-dimensional dynamic culture operation in step (2) includes: The obtained hypothalamic tissue pieces were resuspended in organoid culture medium and seeded into 6-well plates; The cells were placed in a 37°C, 5% CO2 incubator and cultured using an orbital shaker with continuous rotation at 100 rpm.

2. The method for constructing a mouse embryonic hypothalamic organoid model according to claim 1, characterized in that: Step (2) also includes the step of replacing the organoid culture medium: fresh organoid culture medium is replaced every 2 days, and the lysed single cells are removed simultaneously during the replacement to retain the spherical organoid structure.

3. The method for constructing a mouse embryonic hypothalamic organoid model according to claim 2, characterized in that: Step (2) also includes an organoid formation verification step: after 2 weeks of culture, spherical organoid structures with clear boundaries are observed under a microscope.

4. Application of the hypothalamic organoid constructed according to the method according to any one of claims 1 to 3 in neural development research.

5. Application of the hypothalamic organoid constructed according to the method according to any one of claims 1 to 3 in the study of metabolic regulation mechanisms.

6. Use of the hypothalamic organoid constructed according to the method according to any one of claims 1 to 3 in establishing a neuroendocrine disease model.

7. Use of the hypothalamic organoid constructed according to the method according to any one of claims 1 to 3 in establishing a drug screening platform.

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