Construction method of thyroid organoid derived from human induced pluripotent stem cells

Abstract

The invention provides a construction method of a thyroid organoid derived from human induced pluripotent stem cells, and belongs to the technical field of organoid culture. The construction method of the thyroid organ comprises the following steps: (1) separating human induced pluripotent stem cells into single cells, and transferring the single cells to a 2D matrigel working solution for incubation; (2) carrying out endoderm induced culture on the cells incubated in the step (1); (3) carrying out preintestinal endoderm induced culture on the cells obtained in the step (2); (4) carrying out thyroid gland directional induction culture on the cells obtained in the step (3); (5) carrying out thyroid maturation induction culture on the cells obtained in the step (4) to obtain thyroid organs; naI with the final concentration of 0.8-1.2 [mu] M is added during thyroid maturation induction culture. The thyroid organoid constructed by the method is high in maturity, can simulate the state of the in-vivo organoid more truly, and has good capability of secreting thyroid-related hormones at the same time.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to a method for constructing thyroid organoids derived from human induced pluripotent stem cells. Background Technology

[0002] Organoids are cell aggregates formed by the three-dimensional culture of primary tissues or stem cells in vitro. Various cell types can self-organize and self-renew to form complex structures similar to the source tissues or organs, mimicking their physiological functions. Organoids have extremely high application potential in the establishment of thyroid development and disease models. They provide a three-dimensional model for studying thyroid development, compensating for the shortcomings of other biological models.

[0003] Existing experimental protocols for the directed differentiation of functional thyroid organoids using human induced pluripotent stem cells (hiPSCs) include: Figure 1As shown, the specific steps are as follows: Within the first 48 to 72 hours, DE was derived from hiPSCs using the STEMdiff Final Endoderm Kit (a defined, serum-free, animal-component-free medium for differentiating human ESCs and iPSCs into DEs), initiating thyroid differentiation (05110, STEMCELL Technologies). After 48 or 72 hours, DE induction efficiency was assessed by flow cytometry analysis of CKIT and CXCR4. For endoderm precursor cells, cells were embedded at a density of 800–2500 cells / µl in 3D Matrigel (356231, Corning) and cultured for 2 days in cSFDM containing 2 μM dorsomorphin (04-0024, Stemgent), 10 μM SB431542 (1614, Tocris), and 10 μM Y-27632 (1254, Tocris). For thyroid progenitor cell-directed induction, cells were cultured for 10 to 12 days in medium supplemented with 100 ng / ml rhBMP4 (314-BP, R&D Systems), 250 ng / ml rhFGF2 (233-FB, R&D Systems), and 100 ng / ml heparin sodium (HepS) (H4784, Sigma-Aldrich). On the last two days, 10 μM Y-27632 cells were added. Cells were collected from day 14 to day 16 of the total differentiation time, and the expression of NKX2-1 and PAX8 was analyzed. To further differentiate into thyroid cells, the cells were preserved as organoids in a 3D matrix and cultured in cSFDM containing 250 ng / ml rhFGF2, 100 ng / ml rhFGF10 (345FG, R&D Systems), 1 mU / ml bovine TSH (bTSH, Los Angeles Biomedical Research Institute), 50 ng / ml rhIGF-1 (291-G1, R&D Research), 25 ng / ml rhEGF (236EG, R&D Research), 1× insulin-transferrin-selenium (41400-045, Gibco), and 100 ng / ml HepS. 22 days later, 3D Matrigel cells were digested in IMDM (12440, Gibco) medium with 2 mg / ml disase digestive enzyme (17105-041, Gibco). Complete or partially broken cells were removed from 3D Matrigel cells at a ratio of 1:4 at 37°C, and then mature organoids were divided at a ratio of 1:4.Cells were treated with 20 μM PD98059 (9900L, Cell Signaling Technology), 0.1 mM dibutanol-camp sodium salt (114110, Fisher Scientific), and 0.1 mM 3-isobutyl-1-methylxanthine (IBMX; I5897, Sigma-Aldrich) in maturation medium for 3, 6, and 9 days, starting at different times from day 26 to day 41. Cells were collected for further analysis at the end of treatment from day 29 to day 44.

[0004] Although current directed differentiation protocols can generate mature TFCs (thyroid follicular cells), which can present as three-dimensional thyroid follicles in vitro and in vivo, they do not synthesize TH (thyroid hormone) after transplantation in mice. In the scRNA-seq dataset of cells at days 12 and 29, key genes involved in iodine transport, iodine oxidation, TG iodination, TH cleavage, and TH transport were highly expressed in thyroid follicular cells before transplantation; however, the basolateral iodine transporter SLC5A5 (NIS) was expressed only in a few cells.

[0005] Therefore, there is an urgent need in this field to provide a new method for constructing hiPSC-derived functional thyroid organoids, improve the maturity of hiPSC-derived thyroid organoids, ensure good in vivo and in vitro hormone secretion function of thyroid organoids, more realistically simulate the state of in vivo organoids, and provide a better model for in vitro experimental research on thyroid glands. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing thyroid organoids derived from human induced pluripotent stem cells (hiPSCs). Existing literature describes thyroid organoids derived from human induced pluripotent stem cells (hiPSCs) that have been constructed but lack maturity, exhibiting trace amounts of hormones detected in vitro. Furthermore, transplantation of these organoids into hypothyroid mice has shown that thyroid follicles only form in vivo, with no detectable hormone secretion in the blood, and transplantation has not yielded positive results. Considering the deficiencies in existing induction differentiation protocols, a new induction differentiation protocol is proposed to improve the maturity of hiPSC-derived thyroid organoids, ensuring good in vivo and in vitro hormone secretion function, more realistically simulating the in vivo organoid state, and providing a superior model for in vitro thyroid experimental research.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing thyroid organoids derived from human induced pluripotent stem cells, characterized by comprising the following steps: (1) Human induced pluripotent stem cells were separated into single cells and transferred to 2D matrix gel working solution for incubation; (2) The cells incubated in step (1) were subjected to endoderm induction culture; (3) The cells obtained in step (2) were subjected to foregut endoderm induction culture; (4) Perform thyroid-directed induction culture on the cells obtained in step (3); (5) The cells obtained in step (4) are subjected to thyroid maturation induction culture to obtain thyroid organoids; NaI with a final concentration of 0.8~1.2μM is added during the thyroid maturation induction culture.

[0008] Preferably, the degree of cell fusion of the human induced pluripotent stem cells in step (1) is 75-85%; during the separation, the cells are first treated with Gentle Cell dissociation reagent for 6-10 min, and then pipetted 5-10 times in mTeSR medium containing ROCK inhibitor working solution. The 2D matrix gel working solution consists of 2D matrix gel and DMEF / F12 medium; the incubation is carried out at 36~38℃ and 4~6% CO2 for 16~24 hours.

[0009] Preferably, the concentration of the ROCK inhibitor working solution is 8~12μM; the final concentration of the ROCK inhibitor working solution in the mTeSR medium is 0.8~1.2µl / ml; The volume ratio of 2D matrix gel to DMEF / F12 medium in the 2D matrix gel working solution is 200-250 µl: 20-30 ml; the seeding density of single cells on the 2D matrix gel working solution is 1.5 × 10⁻⁶ cells / mL. 6 ~2.0×10 6 1 cell / 6~8ml 2D matrix gel working solution.

[0010] Preferably, the method for endoderm induction culture in step (2) is as follows: After incubation in step (1), the cells were added to STEMdiff™ basal medium containing MR and CJ and cultured for 23-25 ​​hours to obtain cells induced by the first endoderm; the volume ratio of MR, CJ and STEMdiff™ basal medium was 80-120µl: 80-120µl: 8-12ml. The cells induced by the first endoderm induction were cultured in STEMdiff™ shaping endoderm basal medium containing CJ for 23-25 ​​hours to obtain endoderm-induced cells; the volume ratio of CJ to STEMdiff™ shaping endoderm basal medium was 80-120 µl: 8-12 ml.

[0011] Preferably, the method for inducing and culturing foregut endoderm in step (3) is as follows: after lysing the cells, resuspend the cells in 3D matrix gel, and after the 3D matrix gel is solidified, add foregut endoderm induction medium and culture for 40-56 hours. The foregut endoderm induction medium is a completely serum-free differentiation medium containing 1.5-2.5 μM Dorsomorphin, 8-12 μM SB431542, and 8-12 μM ROCK inhibitor.

[0012] Preferably, the method for thyroid-directed induction culture in step (4) is as follows: cells are added to thyroid-specific culture medium and cultured for 8-12 days; The thyroid-specific culture medium is a completely serum-free differentiation medium containing 80-120 ng / ml rhBMP4, 200-300 ng / ml rhFGF2, 80-120 ng / ml Heparin Sodium Salt, and 8-12 μM ROCK inhibitor.

[0013] Preferably, the method for inducing thyroid maturation culture in step (5) is as follows: cells are added to thyroid maturation induction culture medium and cultured for 12 to 30 days; The thyroid maturation induction medium is a completely serum-free differentiation medium containing 80-120 ng / ml rhFGF10, 200-300 ng / ml rhFGF2, 80-120 ng / ml Heparin Sodium Salt, 0.8-1.2 mU / ml bTSH, 20-30 ng / ml hrEGF, 40-60 ng / ml hrIGF-1, 1× Insulin-Transferrin-Selenium, 8-12 μM ROCK inhibitor, and 0.8-1.2 μM NaI.

[0014] Preferably, the serum-free differentiation medium uses Ham's F12 medium and IMDM medium as the basal medium and contains 1× Glutamax, 0.05~0.06% BSA Fraction V, 0.5× B27 Supplement w / o RA, 0.5× N2 Supplement, 0.003~0.005% Monothioglycerol, 40~60 μg / ml Ascorbic acid, and 80~120 μg / ml Primocin at a final concentration; the volume ratio of Ham's F12 medium to IMDM medium is 0.8~1.2:2~4.

[0015] Preferably, the culture is carried out at 36-38°C and 4-6% CO2; the cells are washed with PBS before each culture medium change.

[0016] Preferably, the method further includes the step of identifying thyroid organoids; the identification method includes one or more of the following: transmission electron microscopy, staining analysis, immunofluorescence detection, and detection of thyroid hormone-related hormone secretion capacity. The staining analysis includes HE staining; the immunofluorescence detection is used to detect the expression of thyroid-related proteins; the thyroid hormone-related hormones include thyroxine.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for constructing thyroid organoids derived from human induced pluripotent stem cells. Adding NaI during thyroid maturation induction culture enhances thyroid function and improves the organoid's ability to secrete thyroid hormones. The thyroid organoids constructed using this method exhibit high maturity, more realistically mimicking the in vivo organoid state, and possess excellent ability to secrete thyroid-related hormones. This invention provides a novel and superior model for in vitro thyroid research, possessing practical application value. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 A schematic diagram of the existing hiPSC-induced directed differentiation of functional thyroid organoids; Figure 2 This is a technical roadmap for the method of constructing thyroid organoids derived from human induced pluripotent stem cells in Example 1; Figure 3 The results of observing thyroid organoids under a bright-field microscope in Example 2 are shown in the figure. The scale bar is 50 μm. Figure 4 HE staining results of thyroid organoid sections in Example 2, scale bar is 50 μm; Figure 5 The results of immunofluorescence staining of thyroid organoid sections in Example 2 are shown in the figure. The scale bar is 50 μm. Figure 6 The results of the determination of the ability of thyroid organoids to secrete thyroxine (T4) in Example 2; Figure 7 This is the experimental procedure for the application of thyroid organoids in rats in Example 3. Detailed Implementation

[0020] This invention provides a method for constructing thyroid organoids derived from human induced pluripotent stem cells, characterized by comprising the following steps: (1) Human induced pluripotent stem cells were separated into single cells and transferred to 2D matrix gel working solution for incubation; (2) The cells incubated in step (1) were subjected to endoderm induction culture; (3) The cells obtained in step (2) were subjected to foregut endoderm induction culture; (4) Perform thyroid-directed induction culture on the cells obtained in step (3); (5) The cells obtained in step (4) are subjected to thyroid maturation induction culture to obtain thyroid organoids; NaI with a final concentration of 0.8~1.2μM is added during the thyroid maturation induction culture.

[0021] In this invention, the degree of cell fusion of the human induced pluripotent stem cells in step (1) is 75-85%, preferably 80%; during the separation, the cells are first treated with Gentle Cell dissociation reagent for 6-10 min, preferably 7-9 min, more preferably 8 min, and then pipetted 5-10 times, preferably 7-9 times, more preferably 8 times in mTeSR medium containing ROCK inhibitor working solution. The 2D matrix gel working solution is composed of 2D matrix gel and DMEF / F12 culture medium; the incubation is carried out at 36~38℃ and 4~6% CO2 for 16~24 hours, preferably at 37℃ and 5% CO2 for 20 hours.

[0022] In this invention, the concentration of the ROCK inhibitor working solution is 8-12 μM, preferably 9-11 μM, and more preferably 10 μM; the final concentration of the ROCK inhibitor working solution in the mTeSR medium is 0.8-1.2 µl / ml, preferably 0.9-1.1 µl / ml, and more preferably 1 µl / ml; The volume ratio of 2D matrix gel to DMEF / F12 medium in the 2D matrix gel working solution is 200-250 µl: 20-30 ml, preferably 210-240 µl: 22-28 ml, and more preferably 230 µl: 25 ml; the seeding density of single cells on the 2D matrix gel working solution is 1.5 × 10⁻⁶ cells / day. 6 ~2.0×10 6 1 cell / 6~8ml 2D matrix gel working solution, preferably 1.8×10 6 1 cell / 7ml

[0023] In this invention, the method for endoderm induction culture in step (2) is as follows: The cells incubated in step (1) were added to STEMdiff™ basal medium containing MR and CJ and cultured for 23-25 ​​hours, preferably 24 hours, to obtain cells induced by the first endoderm induction; the volume ratio of MR, CJ and STEMdiff™ basal medium was 80-120µl: 80-120µl: 8-12ml, preferably 90-110µl: 90-110µl: 9-11ml, and more preferably 100µl: 100µl: 10ml; The cells induced by the first endoderm induction are added to STEMdiff™ shaping endoderm basal medium containing CJ and cultured for 23-25 ​​hours, preferably 24 hours, to obtain endoderm-induced cells; the volume ratio of CJ to STEMdiff™ shaping endoderm basal medium is 80-120 μl: 8-12 ml, preferably 90-110 μl: 9-11 ml, and more preferably 100 μl: 10 ml.

[0024] In this invention, the method of foregut endoderm induction culture in step (3) is as follows: after lysing the cells, the cells are resuspended in 3D matrix gel, and after the 3D matrix gel is solidified, foregut endoderm induction culture medium is added and cultured for 40-56 hours. The foregut endoderm induction medium is a completely serum-free differentiation medium containing 1.5-2.5 μM Dorsomorphin, 8-12 μM SB431542, and 8-12 μM ROCK inhibitor; preferably, it is a completely serum-free differentiation medium containing 1.8-2.2 μM Dorsomorphin, 9-11 μM SB431542, and 9-11 μM ROCK inhibitor; more preferably, it is a completely serum-free differentiation medium containing 2 μM Dorsomorphin, 10 μM SB431542, and 10 μM ROCK inhibitor.

[0025] In this invention, the cell lysis method in step (3) is as follows: after washing the cells, mix them with Gentle Cell dissociation reagent and incubate for 4-5 min, preferably 4.5 min, discard the Gentle Cell dissociation reagent and then pipette them with IMDM medium.

[0026] In this invention, the ratio of cells to 3D matrix gel in step (3) is 1.3 × 10⁻⁶. 5 ~1.7×10 5 Cells: 50~70 μl, preferably 1.4 × 10⁻⁶ 5 ~1.6×10 5Cells: 55~65 μl, more preferably 1.5 × 10⁻⁶ μl 5 Cells: 60 μl.

[0027] In this invention, the 3D matrix gel in step (3) is cured by incubating at 36~38℃ and 4~6% CO2 for 15~25 minutes, preferably at 37℃ and 5% CO2 for 20 minutes; the volume ratio of the 3D matrix gel to the foregut endoderm induction medium is 50~70:700~900, preferably 55~65:750~850, and more preferably 60:800.

[0028] In this invention, the method for thyroid-directed induction culture in step (4) is as follows: cells are added to thyroid-specific culture medium and cultured for 8 to 12 days, preferably 9 to 11 days, and more preferably 10 days; The thyroid-specific culture medium is a completely serum-free differentiation medium containing 80-120 ng / ml rhBMP4, 200-300 ng / ml rhFGF2, 80-120 ng / ml Heparin Sodium Salt, and 8-12 μM ROCK inhibitor; preferably, it is a completely serum-free differentiation medium containing 90-110 ng / ml rhBMP4, 220-280 ng / ml rhFGF2, 90-100 ng / ml Heparin Sodium Salt, and 9-11 μM ROCK inhibitor; more preferably, it is a completely serum-free differentiation medium containing 100 ng / ml rhBMP4, 250 ng / ml rhFGF2, 100 ng / ml Heparin Sodium Salt, and 10 μM ROCK inhibitor.

[0029] In this invention, the volume ratio of the thyroid-specific culture medium to the foregut endoderm induction culture medium is 0.8~1.2:0.8~1.2, preferably 0.9~1.1:0.9~1.1, and more preferably 1:1.

[0030] In this invention, the method for inducing thyroid maturation culture in step (5) is as follows: cells are added to thyroid maturation induction culture medium and cultured for 12 to 30 days, preferably 13 to 29 days, more preferably 15 to 25 days, and more preferably 20 days; The thyroid maturation induction medium is a completely serum-free differentiation medium containing 80-120 ng / ml rhFGF10, 200-300 ng / ml rhFGF2, 80-120 ng / ml Heparin Sodium Salt, 0.8-1.2 mU / ml bTSH, 20-30 ng / ml hrEGF, 40-60 ng / ml hrIGF-1, 1× Insulin-Transferrin-Selenium, 8-12 μM ROCK inhibitor, and 0.8-1.2 μM NaI; preferably, it contains 90-110 ng / ml rhFGF10, 220-280 ng / ml rhFGF2, 90-110 ng / ml Heparin Sodium Salt, 0.9-1.1 mU / ml bTSH, 22-28 ng / ml hrEGF, 45-55 ng / ml hrIGF-1, and 1× Insulin-Transferrin-Selenium, 8-12 μM ROCK inhibitor, and 0.8-1.2 μM NaI. A completely serum-free differentiation medium containing Insulin-Transferrin-Selenium, 9-11 μM ROCK inhibitor, and 0.9-1.1 μM NaI; more preferably, a completely serum-free differentiation medium containing 100 ng / ml rhFGF10, 250 ng / ml rhFGF2, 100 ng / ml Heparin Sodium Salt, 1 mU / ml bTSH, 25 ng / ml hrEGF, 50 ng / ml hrIGF-1, 1× Insulin-Transferrin-Selenium, 10 μM ROCK inhibitor, and 1 μM NaI.

[0031] In this invention, the volume ratio of the thyroid maturation induction culture medium to the foregut endoderm induction culture medium is 0.8~1.2:0.8~1.2, preferably 0.9~1.1:0.9~1.1, and more preferably 1:1.

[0032] In this invention, the completely serum-free differentiation medium uses Ham's F12 medium and IMDM medium as the basal medium, and contains 1× Glutamax, 0.05~0.06% BSA Fraction V, 0.5× B27 Supplement w / o RA, 0.5× N2 Supplement, 0.003~0.005% Monothioglycerol, 40~60 μg / ml Ascorbic acid, and 80~120 μg / ml Primocin at a final concentration. Preferably, it contains 1× Glutamax, 0.055~0.058% BSA Fraction V, 0.5× B27 Supplement w / o RA, 0.5× N2 Supplement, 0.0035~0.0045% Monothioglycerol, 45~55 μg / ml Ascorbic acid, and 90~110 μg / ml Primocin at a final concentration. More preferably, it contains 1× The volume ratio of Glutamax, 0.05625% BSA Fraction V, 0.5×B27 Supplement w / o RA, 0.5×N2 Supplement, 0.004% Monothioglycerol, 50 μg / ml Ascorbic acid, 100 μg / ml Primocin; Ham's F12 medium and IMDM medium is 0.8~1.2:2~4, preferably 0.9~1.1:2.5~3.5, and more preferably 1:3.

[0033] In this invention, the culture is carried out at 36-38°C and 4-6% CO2, preferably at 37°C and 5% CO2; the cells are washed with PBS before each culture medium change.

[0034] In this invention, the ROCK inhibitor includes Y-27632.

[0035] This invention also includes a step of identifying thyroid organoids; the identification method includes one or more of the following: transmission electron microscopy observation, staining analysis, immunofluorescence detection, and detection of thyroid hormone-related hormone secretion capacity. The staining analysis includes HE staining; the immunofluorescence detection is used to detect the expression of thyroid-related proteins; the thyroid hormone-related hormones include thyroxine.

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] The reagents used in the following examples are shown in Table 1.

[0038] Table 1. Reagent Names and Sources

[0039] Example 1

[0040] A method for constructing thyroid organoids derived from human induced pluripotent stem cells, the technical roadmap is as follows: Figure 2 As shown, the specific steps are as follows: I. Pre-induction (-1 Day): iPSCs are cultured into single cells. 1. Reagents mTeSR1, Y-27632 (ROCK inhibitor, RI), PBS, Gentle Cell dissociation reagent, trypan blue, 2D matrix gel.

[0041] Preparation of RI working solution: The stock solution concentration used in the experiment is 10 mM. Weigh 1 mg RI and add 310 µl of sterile water to prepare a 10 mM stock solution. Store the stock solution at -20°C and use it within one month (after sterile filtration). Take 1 µl of the stock solution and add 999 µl of water to prepare 1000 µl of a 10 µM working solution. At least 12 ml of culture medium is required for the experiment.

[0042] Preparation of 2D Matrigel working solution: Add 230 µl of 2D Matrigel (hESC-qualified) to 25 ml of DMEF / F12 to prepare 25 ml of base coat working solution. The base coat working solution should not be stored and should be prepared and used immediately. For a 6-well plate (1 ml / well), prepare 7 ml of base coat working solution by adding 64.4 µl of 2D Matrigel to 7 ml of DMEF / F12.

[0043] 2. Experimental Procedure

[0044] (1) Prepare iPSC cells: check for optimal cell confluence (approximately 80%) and remove any spontaneously differentiated cells.

[0045] (2) Separate cells into single-cell suspension in mTeSR + RI working solution (1µl / ml): Take one bottle of T25 human induced pluripotent stem cells and wash with 2ml PBS; incubate with 1 ml Gentle Cell dissociation reagent at RT (room temperature) for 8 minutes; discard the Gentle Cell dissociation reagent; pipette 1ml mTeSR + RI working solution and dissociate the cells into single-cell state 9 times; transfer the cell suspension to a 15ml centrifuge tube and centrifuge at 300 Rcf, 4℃ for 5 min.

[0046] (3) Cell counting and induced pluripotent stem cell plating in single-cell state: according to 1.8×10 6 Cells were resuspended in mTeSR + RI working solution at a density of 10 cells per 6-well plate, then transferred to 2D matrix gel working solution and incubated at 37°C with 5% CO2 for 20 hours.

[0047] II. Day 0: Endoderm Induction - 1

[0048] 1. Reagents

[0049] StemDiff definitive endoderm kit+MR, CJ, PBS.

[0050] Table 2. Composition and dosage of endoderm induction-1 medium

[0051] 1000µl of culture medium: 10 µl of Supplement MR + 10 µl of Supplement CJ + 980 µl of Basal Medium. A total of 12 ml of culture medium is required for the experiment, i.e., 12 µl each of MR and CJ, and 12 ml of basal medium (allit into 6 or 12 ml tubes). The basal medium is STEMdiff™ shaping endoderm basal medium, MR is STEMdiff™ shaping endoderm additive A, and CJ is STEMdiff™ shaping endoderm additive B.

[0052] 2. Experimental Procedure

[0053] Wash cells with 2 ml PBS, add 2 ml basal medium + MR + CJ to each well; incubate at 37°C and 5% CO2 for 24 hours.

[0054] III. Day 1: Endoderm Induction - 2

[0055] 1. Reagents

[0056] StemDiff definitive endoderm kit+CJ, PBS.

[0057] Table 3. Composition and dosage of endoderm induction-2 medium

[0058] Add CJ to cold (2~8℃) STEMdiff™ shaping endoderm basal medium (10µl CJ + 990µl basal medium). A total of 12 ml of medium is required for the experiment, i.e., 12 µl CJ and 12 ml basal medium (dispensed into 6 or 12 ml / tube).

[0059] 2. Experimental Procedure

[0060] Wash cells with 2 ml PBS, add 2 ml StemDiff definitive endoderm kit + CJ to each well; incubate at 37°C and 5% CO2 for 24 hours.

[0061] IV. Day 2: Foregut Endoderm Induction Culture in 3D Matric Gel

[0062] 1. Reagents

[0063] Serum-free differentiation medium (cSFDM), Dorsomorphin, SB 431542, RI, IMDM medium, Gentle Cell dissociation reagent, 3D Matrigel (Matrigel 3D (GFR, phenol red-free Matrigel)) (Corning), 24-well plates, trypan blue staining solution.

[0064] Table 4. Components and dosage of serum-free differentiation medium (cSFDM)

[0065] Preparation of BSA Fraction V: 7.5% = 7.5 g BSA ÷ 100 ml PBS; use 3.75 ml each time, prepare 5 ml at a time, weigh 0.375 g BSA Fraction V to prepare. Store the remaining solution at -20℃ and avoid repeated freeze-thaw cycles.

[0066] Preparation of Ascorbic acid: 500 µl × 10 -3 ×50 mg / ml = 25 mg. Weigh out 25 mg and add it to 500 µl of water.

[0067] Preparation of SB 431542: The experiment requires 5 µl of 10 mM stock solution (store below -20℃ after preparation and use within one month). Weigh 1 mg and add it to 260 µl of DMSO.

[0068] Preparation of Dorsomorphin: Weigh 1 mg of Dorsomorphin and add it to 1.2516 ml of DMSO to prepare a 2 mM stock solution (-80℃, 2 years; -20℃, 1 year, protected from light). Dispense the solution into 100 µl / tubes, avoiding repeated freeze-thaw cycles.

[0069] 2. Experimental Procedure

[0070] (1) Cell lysis

[0071] Wash with 2 ml PBS; incubate with 1 ml Gentle Cell dissociation reagent for 5 minutes at room temperature; discard the Gentle Cell dissociation reagent; pipette the cells with 1 ml IMDM medium; transfer the cell suspension to a centrifuge tube and centrifuge at 300 rcf, 4°C for 5 minutes.

[0072] (2) Cell counting and dispensing

[0073] Aspirate most of the culture medium; in the remaining medium, tap the tube wall to loosen the cells at the bottom; add 60 µl of 3D matrix gel to the 24-well plate and resuspend the cells to a concentration of 1.5 × 10⁻⁶. 5 60µl per cell; incubate at 37°C and 5% CO2 for 20 minutes to solidify the 3D matrix gel; add 800µl of foregut endoderm induction medium (as shown in Table 5) to each well and incubate at 37°C and 5% CO2 for 2 days.

[0074] Table 5. Composition of the foregut endoderm induction medium

[0075] V. Day 4: Thyroid-directed induction - 1

[0076] 1. Reagents

[0077] Thyroid specification media (Table 6), PBS.

[0078] Table 6. Composition of Thyroid specification media

[0079] Preparation of rhBMP4: Reconstitute in 4 mM sterile HCl at a concentration of 100 μg / ml, i.e., 10 μg BMP4 plus 100 µl of 4 mM sterile HCl (after reconstitution, store stably at 2 to 8°C for 1 month under sterile conditions; store stably at -70°C for 3 months).

[0080] Preparation of rhFGF2: Reconstitute in sterile PBS at a concentration of 250 μg / ml, i.e., 10 μg FGF2 added to 40 µl of sterile PBS (after reconstitution, store stably at 2 to 8 °C for 1 month under sterile conditions; store stably at -70 °C for 3 months).

[0081] 2. Experimental Procedure

[0082] Wash cells with 1 ml PBS, add 0.8 ml Thyroid specification media to each well, and incubate at 37°C and 5% CO2 for 2 days.

[0083] VI. Day 6-12: Thyroid-directed induction - 2

[0084] 1. Reagents

[0085] Thyroid specification media (preparation method as above), PBS.

[0086] 2. Experimental Procedure

[0087] Wash cells with 1 ml PBS, add 0.8 ml of Thyroid specification media without added RI to each well, and incubate at 37°C and 5% CO2, changing the medium every 2 days.

[0088] VII. Day 14: Thyroid Maturation - 1

[0089] 1. Reagents

[0090] (1) Thyroid maturation induction medium (Maturation medium + 1 μM NaI), PBS

[0091] Table 7. Components and dosage of Maturation medium

[0092] Preparation of rhFGF10: Reconstitute in sterile PBS at a concentration of 100 μg / ml, i.e., 25 μg rhFGF10 added to 250 µl of sterile PBS (after reconstitution, store stably at 2 to 8°C for 1 month under sterile conditions; store stably at -70°C for 3 months).

[0093] 2. Experimental Procedure

[0094] Wash cells with 1 ml PBS, add 0.8 ml thyroid maturation induction medium to each well, and incubate at 37°C and 5% CO2 for 2 days.

[0095] 8. After Day 16: Thyroid maturation - 2

[0096] As the medium matures, the volume of the medium needs to be adjusted to 1-1.2 ml, or the frequency of changing the medium should be increased to daily, based on the pH change over 48 hours (pH maintained between 7.2 and 7.4).

[0097] 1. Reagents

[0098] Thyroid maturation induction culture medium

[0099] 2. Experimental Procedure

[0100] Wash cells with 1 ml PBS, add 1 ml thyroid maturation induction medium to each well, and incubate at 37°C and 5% CO2 for 13–29 days.

[0101] Example 2: Identification of the thyroid organoid constructed in Example 1

[0102] 1. HE staining

[0103] (1) Organoid agar paraffin embedding

[0104] a. Recover organoids (more than 50 organoids, with an average diameter of 200 μm); b. Remove the original organoid culture medium and add 1 ml of pre-cooled Epithelial Organoid BasalMedium to each well; c. Cut off the tip of the 1ml syringe with scissors; d. Use a 1ml pipette tip rinsed with Anti-Adherence Rinsing Solution (Catalog No.: E238002) to scrape off the matrix gel, causing it to detach. e. Transfer the matrix gel containing organoids into a 1.5 ml EP tube; f. Place the 1.5 ml centrifuge tube on ice and let it stand for 30 minutes to allow the organoids to precipitate naturally; g. Use a 200 µl pipette tip to aspirate and discard as much of the supernatant as possible; (2) Organoid fixation (4% PFA) a. Add 400 µl of 4% paraformaldehyde fixative (PFA), gently pipette to mix the organoids, and incubate at 4°C for 2 hours or at room temperature (RT) for 30 minutes (select the temperature and time according to the density and size of the organoid cells). b. Aspirate 4% PFA, wash organoids 3 times with 1×DPBS, 500 µl each time, gently blow the organoids and let them settle naturally for 5 min, then discard the supernatant. Do not invert the process. c. Place the organoids in the RT container for 10 minutes to allow them to settle naturally, and discard as much of the supernatant as possible; (3) Organoids embedded in 3% low melting point agar a. Weigh 0.3 g of low-melting-point agarose and add it to 9.7 ml of 1×DPBS to completely dissolve the agarose; b. Using a 200 µl pipette tip with the tip cut off, add 140 µl of completely dissolved agarose to the EP tube containing the organoids, so that the agarose completely coats the organoids; c. Place at 4℃ for 5 minutes to allow the agarose to completely solidify, remove the agar block, and trim off the scraps with a scalpel; (4) Dehydration and embedding a. Transfer the agar block to a new EP tube, add 70%, 80%, 90%, and 95% ethanol for 1 hour each for dehydration, and add 100% ethanol for 30 minutes twice. The dehydration process should be carried out on a low-speed horizontal shaker. b. Open the oven about 4 hours in advance, prepare the wax tank, and melt the wax at 65℃; c. After dehydration, transfer the agarose blocks to a tissue embedding cassette (it must be soaked in anhydrous ethanol before xylene treatment). Transfer the embedding cassette to xylene for 5 minutes twice (drain off as much residual xylene as possible each time it is removed; xylene is toxic, this step must be performed in a well-ventilated area). d. Immediately after the transparency treatment is completed, transfer the product to a melted wax tank and immerse it in wax at 60°C for 2 hours. Then, change to a new wax tank and immerse the product for another 2 hours. e. Turn on the embedding machine half an hour in advance. Set the wax tank, wax nozzle, worktable, and left and right preservation boxes to 60°C. Unscrew the wax nozzle and turn on the small cooling stage before embedding. Transfer the tissue embedding box and mold to the melted left and right preservation boxes. Remove the mold and drip in about 25% wax, then place the agar block in the center of the metal mold, and then drip wax for embedding. Remove the tissue embedding box and attach it to the top. After embedding, place it on the freezing stage or in a 4°C refrigerator. After it has completely solidified, carefully remove the mold. (5) Dewaxing of slices a. Fix the embedded paraffin block onto a microtome and cut it into thin sections with a thickness of 5-8 μm. The cut sections are often wrinkled, so they need to be flattened by rinsing them in heated water, then attached to a glass slide, and dried in a 45°C oven. b. Soak the slices in xylene I for 8 min, xylene II for 8 min, anhydrous ethanol I for 1 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, 50% ethanol for 5 min, and distilled water for 5 min in sequence. (6) Staining, dehydration and sealing a. Immerse the sections in Harris hematoxylin staining for 3-8 minutes, then rinse with running tap water for 5 minutes. b. Place the slices in 50% alcohol for 5 min, 70% alcohol for 5 min, 80% alcohol for 5 min, 95% alcohol for 5 min, and anhydrous ethanol for 5 min in sequence; c. Immerse the sections in eosin (lipid-soluble) for 30 seconds, then soak in anhydrous ethanol for 2 minutes; d. Soak in anhydrous ethanol for 1 min, then soak in xylene for 5 min. Remove the sections from the xylene and let them air dry slightly. Mount the sections with neutral resin, let them air dry at room temperature, and then take pictures.

[0105] 2. Immunofluorescence (IF)

[0106] (1) Dewaxing and hydration

[0107] The paraffin slices were baked in a 60℃ constant temperature oven for 30 minutes. Soak the tablets in xylene I for 15 min, then soak them in xylene II for 15 min. Immerse twice in anhydrous ethanol for 5 minutes; twice in 90% ethanol for 5 minutes; twice in 80% ethanol for 5 minutes; twice in 70% ethanol for 5 minutes. Rinse twice in distilled water for 5 minutes each time. (2) Antigen retrieval Place the slides in a sealed slide container and heat the citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) to boiling using a microwave oven. Fill the slide container with the buffer, ensuring all slides are submerged. Place the slide box containing the slides and the buffer solution in an evaporator for 30 minutes (the temperature should be kept close to the boiling point, but not at the boiling point). Remove the slices from the sodium citrate buffer, cool them briefly, and then wash them with 1×PBS buffer for 2 minutes. (3) Staining Rinse the sections with distilled water for 5 min twice; incubate in 3% hydrogen peroxide for 10 min; rinse with distilled water for 5 min twice. The organoid regions were marked using an immunohistochemical pen, and then blocked at room temperature for 2 hours using blocking solution (the blocking solution was serum containing 20% ​​of the same species as the secondary antibody or 5% BSA, with a final concentration of 0.4% Triton X-100 added for membrane perforation). Remove the blocking solution, add an appropriate amount of primary antibody, and incubate overnight at 4°C; Remove the primary antibody diluent, wash with 1×PBS for 3 min, 3 times; Protect from light: Add secondary antibody and incubate at room temperature for 1.5 h; wash with 1×PBS for 3 min, 3 times; Add a drop of DAPI-containing antifluorescence quencher to the slide and gently cover it with a coverslip; Let it stand for 15 minutes, then cover the glass slide with clear nail polish and let it dry for 15 minutes. Observation was performed using a fluorescence microscope. The sources of the primary and secondary antibodies used are shown in Table 8.

[0108] Table 8. Sources of primary and secondary antibodies

[0109] 3. Enzyme-linked immunosorbent assay (ELISA) for detecting in vitro hormone secretion from thyroid organoids.

[0110] 1. Preparation before testing

[0111] (1) Take the kit out of the refrigerator 10 minutes in advance and allow it to equilibrate to room temperature.

[0112] (2) Preparation of standard gradient working solutions: Add 1 ml of universal diluent to the lyophilized standard, let stand for 15 minutes until it is completely dissolved, and then mix gently (concentration is 10 ng / ml). Then dilute according to the following concentrations: 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1.25 ng / ml, 0.625 ng / ml, 0.312 ng / ml, 0.156 ng / ml, 0 ng / ml.

[0113] Dilute using a serial dilution method: Take 7 EP tubes, add 500µl of general diluent to each tube, and pipette 500µl of the 10ng / ml standard working solution into the first EP tube and mix well to prepare a 5ng / ml standard working solution. Repeat this process for the subsequent tubes. The last tube is used as a blank well; it is not necessary to pipette liquid from the second-to-last tube.

[0114] (3) Preparation of Biotin-antibody working solution: 15 minutes before use, centrifuge 100× concentrated Biotin-antibody at 1000×g for 1 minute, and dilute 100× concentrated Biotin-antibody to 1× working concentration with universal diluent.

[0115] (4) Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge 100× concentrated enzyme conjugate at 1000×g for 1 minute, and dilute 100× concentrated enzyme conjugate to 1× working concentration with general diluent.

[0116] (5) Preparation of 1× washing solution: Take 10ml of 20× washing solution into 190ml of distilled water.

[0117] 2. Operating Steps

[0118] (1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining strips in a self-sealing bag and put them back at 4℃.

[0119] (2) Sample addition: Add 50 µl of sample or standard of different concentrations to each well. Add 50 µl of universal diluent to each blank well, followed by 50 µl of Biotin antibody working solution to each well. Cover with sealing film and incubate at 37°C for 60 minutes.

[0120] (3) Washing the plate: Discard the liquid, add 300µl of 1× washing solution to each well, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 3 times.

[0121] (4) Add enzyme conjugate working solution: Add 100µl of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0122] (5) Washing the plate: Discard the liquid and wash the plate 5 times according to the method in step (3).

[0123] (6) Add substrate: Add 90µl of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes.

[0124] (7) Add stop solution: Take out the microplate, add 50µl of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450nm.

[0125] 4. Appraisal Results

[0126] Figure 3 The results of observation of thyroid organoids under a bright-field microscope; Figure 4 HE staining results of thyroid organoid sections show that thyroid organoids possess the conventional morphology of organoids and the follicular structure of thyroid organoids.

[0127] Figure 5Immunofluorescence staining results of thyroid organoid sections show that PAX8 and TTF1 are co-localized in the cell nucleus, providing strong evidence for identifying thyroid organoids. TG is a core marker of thyroid functional maturity; strong positive signals should be concentrated within the lumen of the organoid, mimicking the colloid stored in the follicular lumen of the thyroid gland in vivo. Signals may also be present in the cytoplasm, representing TG synthesis. TSHR is a marker of functional responsiveness; positive signals should be located on the basement membrane side of the cell. ZO-1 is a marker of cell polarity and barrier function, appearing as a clear, continuous linear signal surrounding the lumen edge of each organoid follicle. Ki67 is a marker of proliferative activity; a small number of scattered Ki67-positive cells are present in the image, indicating that the constructed organoid possesses mature function while maintaining self-renewal and growth capabilities, representing an ideal organoid state. Therefore, the hiPSC-derived thyroid organoids constructed in this invention show normal expression of thyroid tissue-related markers, indicating that these organoids possess the ability to secrete thyroid hormones.

[0128] The effect of adding different final concentrations of NaI to the culture medium during the thyroid maturation stage on the ability of thyroid organoids to secrete thyroxine (T4) as follows: Figure 6 As shown in the figure, C is the control (without NaI). It can be seen that the thyroid organoids derived from hiPSC have the ability to secrete T4, and the amount of T4 secreted at the appropriate NaI concentration increased by 50% compared with the control group.

[0129] Example 3

[0130] The thyroid organoids prepared in Example 1 were used in rats. Figure 7 After detecting serum T4 hormone secretion in rats, a rat model of thyroid absence was constructed on September 15, 2025 (method: the thyroid gland was bluntly dissected from the rat's neck). On November 3, 2025, thyroid organoids prepared in Example 1 were transplanted into the model rats. Three weeks after transplantation, blood samples were collected again to detect serum T4 hormone secretion. Serum T4 hormone levels in rats were detected using the Finetest rat T4 detection ELISA kit. The results showed that serum T4 hormone levels were 0 before transplantation, and the T4 concentration in rat serum was 2.99 ng / ml 21 days after transplantation, indicating that transplantation of thyroid organoids may restore thyroid function in rats.

[0131] The procedure for thyroid organoid transplantation is as follows: Prepare the thyroid organoid and resuspend it in PBS; locate the kidney area, open the skin and muscle, gently squeeze out the kidney, and gently lift the kidney capsule with forceps. Make a small incision with a scalpel, attach a syringe tip to a 1ml syringe, aspirate the organoid, and inject it into the kidney capsule through the incision. During the process, continuously moisten the kidney area with physiological saline to prevent it from drying out and cracking. After completion, wait until the organoid is completely adhered to the inside of the kidney capsule, then place the entire kidney back into the abdomen and suture layer by layer.

[0132] 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 method for constructing thyroid organoids derived from human induced pluripotent stem cells, characterized in that, Includes the following steps: (1) Human induced pluripotent stem cells were isolated into single cells and transferred to 2D matrix gel working solution for incubation; (2) The cells incubated in step (1) were subjected to endoderm induction culture; (3) The cells obtained in step (2) were subjected to foregut endoderm induction culture; (4) Perform thyroid-directed induction culture on the cells obtained in step (3); (5) The cells obtained in step (4) are subjected to thyroid maturation induction culture to obtain thyroid organoids; NaI with a final concentration of 0.8~1.2μM is added during the thyroid maturation induction culture.

2. The method according to claim 1, characterized in that, The degree of cell fusion of the human induced pluripotent stem cells in step (1) is 75-85%; during the separation, the cells are first treated with Gentle Cell dissociation reagent for 6-10 min, and then pipetted 5-10 times in mTeSR medium containing ROCK inhibitor working solution. The 2D matrix gel working solution consists of 2D matrix gel and DMEF / F12 medium; the incubation is carried out at 36~38℃ and 4~6% CO2 for 16~24 hours.

3. The method according to claim 2, characterized in that, The concentration of the ROCK inhibitor working solution is 8-12 μM; the final concentration of the ROCK inhibitor working solution in the mTeSR medium is 0.8-1.2 µl / ml; The volume ratio of 2D matrix gel to DMEF / F12 medium in the 2D matrix gel working solution is 200-250 µl: 20-30 ml; the seeding density of single cells on the 2D matrix gel working solution is 1.5 × 10⁻⁶ cells / mL. 6 ~2.0×10 6 1 cell / 6~8ml 2D matrix gel working solution.

4. The method according to claim 1, characterized in that, The method for endoderm induction culture described in step (2) is as follows: After incubation in step (1), the cells were added to STEMdiff™ basal medium containing MR and CJ and cultured for 23-25 ​​hours to obtain cells induced by primary endoderm. The volume ratio of MR, CJ, and STEMdiff™ basal endoderm culture media is 80-120 µl: 80-120 µl: 8-12 ml; The cells induced by the first endoderm induction were cultured in STEMdiff™ shaping endoderm basal medium containing CJ for 23-25 ​​hours to obtain endoderm-induced cells; the volume ratio of CJ to STEMdiff™ shaping endoderm basal medium was 80-120 µl: 8-12 ml.

5. The method according to claim 1, characterized in that, The method for foregut endoderm induction culture in step (3) is as follows: after lysing the cells, resuspend the cells in 3D matrix gel, and after the 3D matrix gel solidifies, add foregut endoderm induction culture medium and culture for 40-56 hours. The foregut endoderm induction medium is a completely serum-free differentiation medium containing 1.5-2.5 μM Dorsomorphin, 8-12 μM SB431542, and 8-12 μM ROCK inhibitor.

6. The method according to claim 1, characterized in that, The method for thyroid-directed induction culture described in step (4) is as follows: cells are added to thyroid-specific culture medium and cultured for 8-12 days; The thyroid-specific culture medium is a completely serum-free differentiation medium containing 80-120 ng / ml rhBMP4, 200-300 ng / ml rhFGF2, 80-120 ng / ml Heparin Sodium Salt, and 8-12 μM ROCK inhibitor.

7. The method according to claim 1, characterized in that, The method for inducing thyroid maturation in step (5) is as follows: add the cells to the thyroid maturation induction medium and culture for 12-30 days; The thyroid maturation induction medium is a completely serum-free differentiation medium containing 80-120 ng / ml rhFGF10, 200-300 ng / ml rhFGF2, 80-120 ng / ml Heparin Sodium Salt, 0.8-1.2 mU / ml bTSH, 20-30 ng / ml hrEGF, 40-60 ng / ml hrIGF-1, 1× Insulin-Transferrin-Selenium, 8-12 μM ROCK inhibitor, and 0.8-1.2 μM NaI.

8. The method according to any one of claims 5 to 7, characterized in that, The serum-free differentiation medium used Ham's F12 medium and IMDM medium as basal media, and contained 1× Glutamax, 0.05~0.06% BSA Fraction V, 0.5× B27 Supplement w / o RA, 0.5× N2 Supplement, 0.003~0.005% Monothioglycerol, 40~60μg / ml Ascorbic acid, and 80~120μg / ml Primocin; the volume ratio of Ham's F12 medium to IMDM medium was 0.8~1.2:2~4.

9. The method according to any one of claims 1 to 7, characterized in that, All cultures were conducted at 36-38°C and 4-6% CO2; cells were washed with PBS before each culture medium change.

10. The method according to claim 1, characterized in that, It also includes the step of identifying thyroid organoids; the identification methods include one or more of the following: transmission electron microscopy, staining analysis, immunofluorescence detection, and detection of thyroid hormone-related hormone secretion capacity; The staining analysis includes HE staining; The immunofluorescence assay is used to detect the expression of thyroid-related proteins; the thyroid hormone-related hormones include thyroxine.

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