Cell-proliferation adrenocortical organoid produced using adrenal cortex-derived cells, cell-based pharmaceutical containing adrenocortical organoid, and method for producing adrenocortical organoid

Abstract

The present invention relates to: an adrenocortical organoid produced using adrenal cortex-derived cells; a cell-based pharmaceutical containing the adrenocortical organoid; and a method for producing the adrenocortical organoid.

Description

Cell-proliferative adrenal cortical organoids produced using adrenal cortical-derived cells, cell-based pharmaceutical products containing said adrenal cortical organoids, and methods for producing said adrenal cortical organoids.

[0001] The present invention relates to a cell-proliferative adrenocortical organoid produced using adrenocortical-derived cells, a cell-based pharmaceutical product containing the adrenocortical organoid, and a method for producing the adrenocortical organoid.

[0002] The adrenal glands are organs that produce adrenocortical hormones. Stimulation from the hypothalamus with corticotropin-releasing hormone (CRH) increases the secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. The secreted ACTH stimulates the adrenal cortex, leading to the production of adrenocortical hormones (Lyraki, 2021, Nat Rev Endocrinol, doi: 10.1038 / s41574-021-00491-4). These three components regulate each other through a feedback loop, and this regulatory system is called the hypothalamic-pituitary-adrenal axis. Adrenal cortical hormones are important hormones in the body involved in energy production, inflammation, and stress response. Among them, cortisol is a very important hormone involved in maintaining life.

[0003] Adrenal insufficiency is a condition in which adrenal cortical cells fail to produce adrenal cortical hormones for various reasons and is life-threatening. Adrenal insufficiency is known to be caused by autoimmune diseases, infectious diseases, heredity, metabolic disorders, tumors, etc. (Husebye, 2021, Lancet, DOI: 10.1016 / S0140-6736(21)00136-7). In bilateral adrenal tumors, the adrenal tissues on both sides are surgically removed, resulting in the inability to produce adrenal cortical hormones in the body. As a result, patients need to take hormone agents (hormone replacement) throughout their lives. So far, the treatment of adrenal insufficiency has been limited to hormone replacement therapy. For example, cortisol that is deficient in the body due to adrenalectomy can be supplemented by taking hydrocortisone. In the natural state, the hypothalamic-pituitary-adrenal axis secretes the required amount of adrenal cortical hormones in the body according to the circadian rhythm and stress. However, in hormone replacement therapy, due to the absence of the hypothalamic-pituitary-adrenal axis, there is a deviation between the required amount of adrenal cortical hormones in the body and the amount of hormones ingested, and various complications (side effects) such as fatigue, difficulty working, metabolic disorders, hypercholesterolemia, or lifestyle diseases are likely to occur. Also, the risk of forgetting to take the medicine has been a problem.

[0004] Parathyroid transplantation can be cited as a technique established for the transplantation treatment of endocrine glands. As a treatment for hyperparathyroidism, the parathyroid gland is removed and a part of it is transplanted into the muscle of the arm (Non-Patent Document 1). Similarly, for bilateral adrenal insufficiency, a treatment method in which the cortex is separated from the removed adrenal gland, minced into 1 mm squares, and transplanted into the muscle of the arm has been studied, but there are reports of good and poor tissue engraftment after transplantation, and stable results have not been obtained (Non-Patent Document 2). A method for producing adrenal cortical-like cells from human iPS cells has been reported. In the method of forcibly expressing the NR5A1 gene, Δ4-steroid-producing cells such as cortisol and aldosterone are produced (Non-Patent Document 3). In the method of differentiating through the posterior intermediate mesoderm and adrenal primordium, Δ5-steroid-producing cells such as DHEA and DHEA-S are produced (Non-Patent Document 4).

[0005] Wells, 1975, Surgery, PMID: 1138398Dubernard, 1995, Transplant Proc., PMID: 7878899 / Grodstein, 2010, Am. J. Transplant., DOI: 10.1111 / j.1600-6143.2009.02929.xYazawa, 2009 Endocrinology, DOI: 10.1210 / en.2008-1310 / Sonoyama, 2012, Endocrinology, DOI: 10.1210 / en.2012-1060 / Li, 2019, PNAS, DOI: 10.1073 / pnas.1908207116Sakata, 2022, Dev. Cell, DOI: 10.1016 / j.devcel.2022.10.010

[0006] The objective of this invention is to provide novel adrenal cortical organoids (hAGOs) produced using adrenal cortex-derived cells.

[0007] As a result of diligent research, the inventors have discovered that a novel adrenal cortical organoid created using adrenal cortical-derived cells can reconstruct the hypothalamus-pituitary-adrenal axis in vivo, thus completing the present invention. That is, the present invention includes the following aspects.

[0008] Item 1. A cell-proliferative adrenocortical organoid obtained by three-dimensional culture of adrenocortical-derived cells. Item 2. A cell-based drug comprising the adrenocortical organoid of Item 1. Item 3. The cell-based drug according to Item 2, which is a cell-based drug for the treatment of adrenal insufficiency. Item 4. The cell-based drug according to Item 2 or 3, wherein the adrenal insufficiency is at least one type of adrenal insufficiency selected from the group consisting of bilateral pheochromocytoma, Addison's disease (subsequent adrenocortical hormone deficiency), and side effects of anticancer treatment with immune checkpoint inhibitors (ICIs). Item 5. The cell-based drug according to any one of Items 2 to 4, wherein the adrenocortical-derived cells are autologous cells. Item 6. The adrenocortical organoid according to Item 1 or the cell-based drug according to any one of Items 2 to 5, wherein the adrenocortical organoid expresses at least one marker selected from the group consisting of CYP17A1, CYP11B1, CYP11B2, HSD3B, and KCNJ. Item 7. (A) A method for producing adrenal cortical organoids, comprising the step of three-dimensionally culturing adrenal cortical-derived cells in a medium containing at least one of WNT and a substance that activates the WNT pathway, at least one of adrenocorticotropic hormone (ACTH) and a substance that activates the ACTH pathway, and at least one of Sonic Hedgehog (SHH) and a substance that activates the SSH pathway. Item 8. The method for producing adrenal cortical organoids according to item 7, further comprising step (B): (B) Further three-dimensionally culturing the cells obtained in step (A) in a medium containing retinoic acid (RA). Item 9. A method for producing a drug according to claim 7 or 8, comprising: (A') three-dimensionally culturing adrenocortical-derived cells in a medium containing at least one of WNT and a substance that activates the WNT pathway, at least one of ACTH and a substance that activates the ACTH pathway, at least one of SHH and a substance that activates the SSH pathway, EGF, FGF10, FGF7, and FGF2; and (B') further three-dimensionally culturing the cells obtained in step (A') in a medium containing oncostatin M (OSM) and RA. Claim 10. Use of adrenocortical organoids as cell-based pharmaceuticals.

[0009] According to the present invention, a novel adrenal cortical organoid is provided, which is prepared using adrenal cortical-derived cells.

[0010] This is a schematic diagram showing the reconstruction of the thalamic-pituitary-adrenal axis using adrenal organoids (hAGOs). The diagrams show the time course of hAGO culture, HE staining of hAGOs and human adrenal cortex, and secretory granules in hAGOs and mouse adrenal cortical cells. The diagrams show the expression of adrenal cortical markers in hAGOs. The diagram confirms cortisol production in mice transplanted with hAGOs. The diagram confirms the regeneration of adrenal tissue after transplantation of the prepared hAGOs into mice. The diagram confirms the presence of adrenal cortical stem cells and adrenal cortical cells in hAGOs. The diagram examines the cell proliferation of hAGOs. The diagrams show the effect of hAGO transplantation on the survival time of adrenalectomized mice.

[0011] The adrenal cortical organoid (hereinafter referred to as hAGO) of the present invention is obtained by collecting adrenal cortical-derived cells and then culturing the cells in three dimensions using an extracellular matrix gel or a non-adherent plate. The adrenal cortical-derived cells include primary cells collected from adrenal tissue and cells obtained by subculturing, expanding, etc. The adrenal cortical-derived cells of the present invention are obtained by isolating them from adrenal tissue collected from a living organism. The adrenal tissue may be obtained from either a healthy person or / or adrenal tissue excised from a patient with adrenal insufficiency. For example, if hAGO is produced using adrenal cortical-derived cells collected from a patient with adrenal insufficiency, the produced hAGO will be autotransplanted, which is preferable because it avoids immunological problems. In other words, it is preferable that the hAGO in the present invention is composed of autologous cells.

[0012] For the creation of hAGOs, it is conceivable to use adrenocortical cells obtained by differentiation induction from, for example, ES cells or iPS cells. In this case, the iPS cells used are generally those stored in iPS cell banks, etc., and collected from a donor different from the recipient of the hAGO transplant. From these, cells that provide a certain degree of immune tolerance are selected and used, but complete immune tolerance cannot be achieved. Therefore, when transplanting hAGOs using iPS cell-derived adrenocortical cells, it is thought that immunosuppressants must be used compared to when autologous cells are used. Furthermore, if iPS cells derived from the recipient of the hAGO transplant are to be used in the first place, the iPS cells must be created. Moreover, adrenocortical cells obtained by differentiation induction from iPS cells are genetically modified cells or can only produce hormone precursors, and at present there are high barriers to practical application. For these reasons, the transplantation of hAGOs using autologous cells is preferable because it does not require the use of immunosuppressants and does not incur the cost of creating iPS cells derived from the recipient patient. In other words, the hAGO of the present invention is an organoid produced using cells obtained from adrenal tissue collected from a living organism, and does not include organoids produced using adrenal cortical cells obtained by differentiation induction from ES cells or iPS cells.

[0013] The hAGO of the present invention is a cell-proliferating organoid. Because the hAGO of the present invention contains tissue stem cells, it exhibits cell-proliferating properties. WNT signaling and SHH signaling are induced in the culture medium of the organoid, recreating the stem cell niche and allowing the stem cells to proliferate. The proliferated stem cells differentiate into adrenocortical cells and secrete adrenocortical hormones such as cortisol. By transplanting an organoid containing stem cells and differentiated cells, cell turnover can be recreated in the body, and the tissue can be maintained for a long period of time, similar to the original tissue. For this reason, it is important to transplant an organoid with a cell number comparable to that of the harvested tissue. As described above, since the hAGO of the present invention is a cell-proliferating organoid, it is advantageous that the cells can be proliferated in vitro before transplantation. In the cell-proliferating hAGO of the present invention, the proportion of stem cells in the total cells forming the hAGO is preferably 20% to 70%, more preferably 20% to 60%, and most preferably 25% to 40%. By having the stem cell proportion at the lower limit of the above, a cell-proliferating hAGO can be obtained. Because the proportion of stem cells is at the above upper limit, the resulting hAGO has the function of reconstructing the hypothalamic-pituitary-adrenal axis in vivo.

[0014] The present invention provides a method for producing hAGO, comprising step (A) of three-dimensional culture of adrenocortical cells in a medium containing at least one of WNT and a substance that activates the WNT pathway, at least one of adrenocorticotropic hormone (ACTH) and a substance that activates the ACTH pathway, and at least one of Sonic Hedgehog (SHH) and a substance that activates the SSH pathway. The medium used in step (A) may further contain at least one substance selected from the group consisting of EGF, FGF10, FGF7, and FGF2, and more preferably contains EGF, FGF10, FGF7, and FGF2 (the medium used in step (A')). The medium used in the method for producing hAGO of the present invention may contain, for example, WNT3A as the WNT, and may contain, for example, R-spondin as the substance that activates the WNT pathway, or may contain both WNT and R-spondin. The culture medium used in the method for producing hAGO of the present invention may contain ACTH, and may also contain a substance that activates the ACTH pathway, such as cosyntropin. More preferably, the culture medium contains ACTH or a substance that activates the ACTH pathway. The culture medium used in the method for producing hAGO of the present invention may contain SHH, and may also contain a substance that activates the SHH pathway, such as purmorphamine. More preferably, the culture medium contains SHH or a substance that activates the SHH pathway. These WNT and substances that activate the WNT pathway, ACTH and substances that activate the ACTH pathway, and SHH and substances that activate the SHH pathway may each be used individually or in combination of two or more substances. The method for producing hAGO of the present invention preferably includes a step (B) in which the cells obtained in step (A) are further cultured in three dimensions in a medium containing retinoic acid (RA), and more preferably a step (B') in which the cells are further cultured in three dimensions in a medium containing oncostatin M (OSM) in addition to RA.The method of three-dimensional culture is not particularly limited, but it can be cultured according to existing methods such as culturing on low-adhesion plates or in matrix gels (Sato, 2009, Nature, DOI: 10.1038 / nature07935), for example, as follows: The adrenal cortex is separated from the adrenal tissue, cells are collected by enzymatic treatment with collagenase, etc., and the collected cells are cultured in a medium suitable for adrenal cortical cells to obtain primary cells. At that time, the sonic hedgehog signal is induced using Purmorphamine or SHH, and the WNT signal is induced using WNT3A and R-spondin. The obtained cultured cells can be cultured in three dimensions by culturing them in Matrigel. As shown in the examples described later, the researchers have established a method for culturing hAGO by excising the cortex from the adrenal gland resected from a pheochromocytoma patient.

[0015] Examples of gels used for the three-dimensional culture of hAGO in this invention include Matrigel (Corning International), which contains extracellular matrix proteins; Cellmatrix (Nitta Gelatin Co., Ltd.), a collagen gel; and GrowDex (UPM Biomedicals, Finland), a cellulose gel.

[0016] The cell-based pharmaceutical product of the present invention contains hAGO. By administering the cell-based pharmaceutical product of the present invention to humans, adrenal insufficiency can be treated. First, the hAGO contained in the cell-based pharmaceutical product of the present invention engrafts at the transplantation site in the body. As a result of stimulation of CRH in the hypothalamus, ACTH secreted from the anterior pituitary gland acts on the engrafted hAGO through the bloodstream, causing the hAGO to produce adrenocortical hormones. The secreted adrenocortical hormones act on the hypothalamus and pituitary gland through the bloodstream. Such a feedback control mechanism (hereinafter referred to as the hypothalamic-pituitary-adrenal axis in this specification) operates between the hypothalamus, pituitary gland, and hAGO. Therefore, it is possible to reconstruct the hypothalamic-pituitary-adrenal axis regardless of where the hAGO is transplanted in the body. In this way, the cell-based pharmaceutical product of the present invention, which can reconstruct the hypothalamic-pituitary-adrenal axis, can achieve cortisol secretion in accordance with the body's needs and can reduce the possibility of side effects, thus being safer than existing hormone replacement therapy. Furthermore, while hormone therapy carries risks such as forgetting to take medication, the hAGO transplantation method of this invention is advantageous because it can free patients from the obligation of taking medication. Moreover, since a single hAGO transplantation can maintain the recovery of adrenal function over the long term, it contributes to improving the patient's quality of life.

[0017] The site in which the hAGO of the present invention is transplanted into a human is not particularly limited and may be anywhere in the body. When selecting a transplant site, a site with a high engraftment rate and hormone secretion rate, and low invasiveness, is preferred. Possible transplant sites include the thigh muscle, subcapsular renal capsule, and subcutaneous tissue. Among these, subcutaneous transplantation is preferred to reduce the burden on the patient as it is less invasive. Transplanting to a less invasive site can reduce the risk of complications in the patient after transplantation and improve the quality of life after surgery.

[0018] The appropriate amount of hAGO transplanted in humans may be determined, for example, from the amount of adrenocortical hormone secreted or from information extrapolated from dose experiments conducted in clinical trials. The amount of hAGO can be quantified from in vitro culture and in vivo animal experiments and used to calculate the appropriate dosage. In this invention, it is preferable to transplant about 5 grams of hAGO into humans. If a patient who has undergone partial adrenalectomy does not have 5 grams or more of adrenal cortex remaining in the body, hormone replacement therapy will be necessary. Therefore, the same amount is considered to be a reference when treating adrenal insufficiency without hormone replacement therapy by transplanting the hAGO of this invention. Since the adrenal cortex contains cells other than secretory cells, the required cell amount is thought to be 5 grams or less.

[0019] To obtain approximately 5 grams of hAGO, methods for mass culture of adrenocortical cells include suspension culture and culture using low-adhesion plates. Culture in a medium containing an extracellular matrix is ​​also possible. Examples of such extracellular matrices include Matrigel, collagen, and cellulose. For example, Cellumatrix (Nitta Gelatin Co., Ltd.) can be used as a collagen gel, and GrowDex (UPM Biomedicals, Finland) as a cellulose gel.

[0020] The cell therapy of the present invention may be a composition comprising, in addition to the hAGO, one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. The composition may include an extracellular matrix gel (such as Matrigel, collagen, or cellulose), a buffer (such as neutral buffered saline or phosphate-buffered saline), carbohydrates (such as glucose, mannose, dextran, starch, lactose, sucrose, malt, rice, or wheat flour), sugar alcohols (such as sorbitol, mannitol, xylitol, erythritol, or maltitol), serum albumin, gelatin, proteins (such as immunoglobulins), amino acids (such as polypeptides or glycine), chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, or dried skimmium. The following may be included, but are not limited to, glycerol, propylene glycol, ethanol, gum arabic, alginate, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, hydrophilic polymers (such as polyvinylpyrrolidone), TWEEN®, polyethylene glycol, water, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, antioxidants, chelating agents (such as EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), solubilizers, emulsifiers, preservatives, and other similar agents.

[0021] The cell-based therapeutic agent of the present invention can be administered in combination with other therapeutic agents. In this case, the cell-based therapeutic agent of the present invention and the other therapeutic agents can be administered simultaneously, sequentially, or individually. The other therapeutic agents may be, but are not limited to, drugs such as compounds or proteins that have preventive, therapeutic, and ameliorative effects on adrenal insufficiency.

[0022] Adrenal insufficiency in this invention includes bilateral pheochromocytoma, congenital adrenal hypoplasia, Addison's disease (later-onset adrenocortical hormone deficiency), or adrenal insufficiency as a side effect of anticancer treatment with immune checkpoint inhibitors (ICIs). By transplanting the hAGO according to this invention, the hypothalamic-pituitary-adrenal axis can be reconstructed in the body, thereby enabling the treatment of these adrenal insufficiencies.

[0023] Bilateral pheochromocytoma is a tumor of the adrenal medulla. Although the cortex is normal, both adrenal glands are removed, resulting in a disease in which the body is unable to produce adrenal hormones. Addison's disease is a disease in which the production of adrenocortical hormones decreases acquired later in life (posterior adrenocortical dysplasia). Other conditions include adrenal insufficiency in patients who have undergone anticancer treatment with immune checkpoint inhibitors (ICIs) as a side effect. By transplanting the hAGO of the present invention into patients who have developed adrenal insufficiency due to these diseases, the hypothalamic-pituitary-adrenal axis can be reconstructed in the body, thus safely treating adrenal insufficiency.

[0024] The hAGO of the present invention produces adrenal hormones similar to those found in the adrenal glands in the body. Examples of such hormones include cortisol, aldosterone, and androgens (DHEA, DHEA-S).

[0025] The hAGO of the present invention expresses at least one marker selected from the group consisting of CYP17A1, CYP11B1, CYP11B2, HSD3B, and KCNJ. CYP11B1 is a protein involved in cortisol production. CYP11B2 is a marker involved in aldosterone production. CYP17A1 is expressed in enzymes that produce precursors of cortisol and aldosterone, and is involved in the production of adrenal cortical hormones in general. HSD3B is involved in the production of cortisol, aldosterone, and androgens. KCNJ is a marker gene for the zona glomerulosa of the adrenal cortex.

[0026] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations.

[0027] Experiment 1. Production of Adrenal Cortical Organoids (hAGOs) The cortex was excised from the adrenal gland of a pheochromocytoma patient. Next, adrenal cortical cells were collected from the cortex by enzymatic treatment with collagenase, suspended in Matrigel, and placed on a plate. After the Matrigel solidified, DMEM / F12 medium supplemented with growth factors (WNT3A, R-spondin, EGF, FGF10, FGF7, FGF2, Purmorphamine, ACTH) was added, and the cells were cultured for 7 days at 37°C and 5% CO2 to obtain organoids (hAGOs). The culture results are shown in Figure 2.

[0028] As shown in Figure 2a, hAGO was formed in about one week. Its interior was filled with vesicles (hAGO in Figure 2b). Furthermore, secretory vesicles were detected in the hAGO using a holotomography microscope (Figure 2c). Holotomography microscopy revealed white granules in adrenal cortical cells, suggesting the accumulation of secretory granules within the cells. As mentioned above, similar images were obtained in the hAGO, suggesting that the hAGO of the present invention possesses characteristics of endocrine cells. Adrenal tissue in vivo is known to have different morphologies called the zona glomerulosa (ZG in Figure 2b), the zona fasciculata (ZF in Figure 2b), and the zona reticularis (ZR in Figure 2b). The HE-stained image of the hAGO (hAGO in Figure 2b) was morphologically similar to the zona reticularis (ZR in Figure 2). Furthermore, it was confirmed that the created hAGO expressed CYP17A1, which is involved in the production of adrenocortical hormones in general; CYP11B1, which is involved in cortisol production; CYP11B2, which is involved in aldosterone production; HSD3B, which is involved in cortisol, aldosterone, and androgen production; KCNJ, a marker protein of the adrenal zona glomerulosa; CDH2, a marker for the entire adrenal cortex; NESTIN, a marker for adrenal cortical stem cells; DHH, etc. (Figure 3)

[0029] Next, the prepared hAGO was stimulated with ACTH (3 nM), an adrenocorticotropic hormone, and the cortisol concentration secreted into the culture medium was measured, confirming a significant increase with the addition of ACTH (Figure 4a). Human adrenal cell line (H295R cells) was used as a positive control, and organoids prepared using cells collected from adrenal tumors (Tumor-AGO) were used as a negative control. As a result, it was confirmed that a significant amount of cortisol was produced from the prepared hAGO (Figure 4b). These results clearly show that the hAGO of the present invention, like those in the human body, exhibits improved hormone production, including cortisol, upon ACTH stimulation.

[0030] Experiment 2. Transplantation of hAGO under the renal capsule of immunodeficient mice. hAGO obtained in Experiment 1 was transplanted under the renal capsule of immunodeficient mice. After 30 days, the transplanted hAGO was excised and stained with HE (Figure 5a), and it was confirmed that tissue similar to that of the adrenal gland had formed (Figure 5b). When the excised hAGO-derived tissue was magnified (Figure 5c), a morphology similar to the zona glomerulosa (ZG in Figure 2b), which produces aldosterone, and the zona fasciculata (ZF in Figure 2b), which produces cortisol, was observed. Thus, it was revealed that when the prepared hAGO was transplanted into mice, adrenal tissue was formed in vivo in the mice.

[0031] Experiment 3. Transplantation of hAGO into the thigh muscle of immunodeficient mice. hAGO obtained in Experiment 1 was transplanted into the thigh muscle of immunodeficient mice (nude mice) and nude mice from which the adrenal glands had been removed. Cortisol was detected in the blood (serum) of these mice for 50 days after transplantation (Figure 4c: nude mouse and Figure 4d: nude mouse from which the adrenal glands had been removed). The adrenal cortical hormone in mice is not cortisol, but its precursor, corticosterone, and normally cortisol is not present. Therefore, it can be said that the cortisol detected in this experiment was produced by the transplanted hAGO. The human blood cortisol concentration is known to be around 0.1-0.2 μg / mL. Since the hAGO of the present invention produces cortisol at a level similar to that of humans in the body of mice, it is expected that the hAGO of the present invention can function sufficiently in the human body as well. Furthermore, in transplants into immunodeficient mice that underwent bilateral adrenalectomy, cortisol secretion into the bloodstream was detected more rapidly compared to non-adrenalized immunodeficient mice (Figure 4c) (Figure 4d). This is thought to be because adrenalectomy increased ACTH secretion, which in turn promoted the growth of the transplanted cells.

[0032] As described above, the hAGO of the present invention was confirmed to engraft and exert adrenal function after being transplanted into the thigh muscle and renal capsule of immunodeficient mice. Previously, for parathyroid tumors in type 1 multiple endocrine neoplasia, the treatment method of transplanting non-tumor tissue into the forearm muscle has been chosen. However, in adrenal tumors, it is known that transplanted tissue from the non-tumor portion of the adrenal cortex is difficult to engraft. The present invention is expected to be extremely useful as it can provide a new treatment method for diseases that were difficult to treat with conventional methods.

[0033] Experiment 4. Confirmation of the presence of adrenal cortical cells in hAGO (spatial transcriptome analysis) The hAGO created in Experiment 1 and the hAGO transplanted into mice in Experiment 2 were excised, and organoids (KC-hAGO) created again using the excised cells, along with human adrenal tissue, were embedded in gel, and gene expression in each region was examined by spatial transcriptome analysis. For detection, markers expressed in stem cells and differentiated cells respectively are used (adrenocortical stem cell markers: PTCH1, PTCH2, SMO, GLI1, GLI2, GLI3, RPO3, WNT2B, FZD1, FZD2, LRP1, WNT5B, WNT9A, HES4, JAG1, BMP3, FGFR1, HOPX, HOXC4, KLF2, KLF5, KLF9, JUN, LRIG1, LRIG3, NFIX, NR2F”, NR2F1, NRC3, POU6F1, SRF, TBX2, TBX18, TCF21, TCF7L1; adrenocortical differentiation markers: CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP21A2, HSD3B2, HSD3B7, KCNJ5, KCNJ8, CYP51A1, FGF9, and FGF12 were used. The results are shown in Figure 6.

[0034] Figure 6a shows human adrenal cortical tissue on the right and hAGO on the left. The hAGO is solidified into a block using alginate gel, and a thin section of this block is shown in Figure 6a (left). hAGO and KC-hAGO are embedded side by side in this thin section. A plot of human adrenal cortical tissue (Figure 6a (right)) shows that stem cells are present in the adrenal capsule region and differentiated cells are present in the adrenal cortical region, and it was found that detection with the marker can correctly classify stem cells and differentiated cells in adrenal tissue (Figure 6b). Next, as shown in Figure 6a (left), it became clear that hAGO and KC-hAGO also contain cells that can be classified as stem cells and differentiated cells, respectively. These organoid-derived cells were classified into different clusters than those in human adrenal tissue (Figure 6b). The proportions of stem cells, differentiated cells, and other cells differed among human adrenal tissue, hAGO, and KC-hAGO (Figure 6c). The higher proportion of stem cells in hAGO and KC-hAGO compared to human adrenal tissue suggests that these culture conditions support stem cell proliferation (Figure 6c). Furthermore, it is thought that cells differentiated from stem cells into adrenal cortical cells (Figure 6d-f) secreted cortisol, as shown in Figure 4. The results in Figure 6c suggest that if the proportion of stem cells in the total cells forming hAGO is between 20% and 70%, a proliferative hAGO with the function of reconstructing the hypothalamic-pituitary-adrenal axis in vivo can be obtained.

[0035] Next, excluding stem cells, we performed expression analysis on differentiated adrenal cortical cells only (Figure 6d-f), dividing them into three regions: zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). In human adrenal tissue, as previously known, ZG, ZF, and ZR were shown to be present in layers within the adrenal cortex. In hAGO and KC-hAGO, cells classified as ZG, ZF, and ZR were also found. When we examined the proportion of cells classified as ZG, ZF, and ZR (Figure 6f), similar to the results in Figure 6c, the proportion of cells classified as "other" was also high in organoids, meaning that cells in the process of differentiation were included, suggesting that the stem cell-differentiated cell cycle is maintained.

[0036] Experiment 5. Cell Proliferation Experiment 1: The hAGOs prepared in Experiment 1 were cultured in media containing various factors, and the organoid proliferation was investigated. A base medium containing WNT, R-spondin, Noggin, FGF10, FGF7, FGF2, Insulin, Transferrin, Selenium, FBS, and HS was used, and the proliferation was compared when a GSK-3 inhibitor (CHIR), retinoic acid (RA), Notch activator (CPA), or a combination thereof was added to the base medium. Four hAGOs were cultured under each condition, and the lengths of the short and long sides (μm) of each hAGO were measured, and the volume of each hAGO was estimated from these values. The results are shown in Figure 7.

[0037] Figure 7a shows representative images of hAGOs after 14 days of culture at 37°C and 5% CO2 in media supplemented with each factor, both in the presence and absence of oncostatin M (OSM). It was found that hAGOs cultured in media supplemented with RA showed significantly greater organoid proliferation compared to other conditions. Figure 7b shows the estimated volume of hAGOs after 14 days of culture under each condition. Figure 7b also shows that significantly greater hAGO proliferation was observed under the condition of RA supplementation. From these results, it was found that hAGOs with higher cell proliferation can be obtained by further three-dimensional culture, especially in media containing RA.

[0038] Experiment 6. Similar to Experiment 3, which involved functional complementation by hAGO transplantation in adrenalectomized mice, the adrenal glands were removed from nude mice. The survival times of the group that received hAGO transplantation in Experiment 1 (N=4) and the group that did not receive hAGO transplantation (N=4) were analyzed (Figure 8). All non-transplanted adrenalectomized mice died within 70 days. This confirmed that hAGO transplantation complements adrenal function in adrenalectomized mice.

Claims

1. Cell proliferation-proliferating adrenal cortical organoids obtained by three-dimensional culture of adrenal cortical-derived cells.

2. A cell-based pharmaceutical product comprising the adrenal cortical organoid of claim 1.

3. The cell-based pharmaceutical product according to claim 2, which is a cell-based pharmaceutical product for the treatment of adrenal insufficiency.

4. The cell-based pharmaceutical product according to claim 3, wherein the adrenal insufficiency is at least one type of adrenal insufficiency selected from the group consisting of bilateral pheochromocytoma, Addison's disease (degenerative adrenocortical hormone deficiency), and side effects of anticancer treatment with immune checkpoint inhibitors (ICIs).

5. The cell-based pharmaceutical product according to claim 2, wherein the adrenal cortex-derived cells are autologous cells.

6. The adrenal cortical organoid according to claim 1 or the cell drug according to claim 2, wherein the adrenal cortical organoid expresses at least one marker selected from the group consisting of CYP17A1, CYP11B1, CYP11B2, HSD3B, and KCNJ.

7. (A) A method for producing adrenal cortical organoids, comprising the step of three-dimensionally culturing adrenal cortical-derived cells in a medium containing at least one of WNT and a substance that activates the WNT pathway, at least one of adrenocorticotropic hormone (ACTH) and a substance that activates the ACTH pathway, and at least one of Sonic Hedgehog (SHH) and a substance that activates the SSH pathway.

8. The method of preparation according to claim 7, further comprising step (B): (B) a step of further culturing the cells obtained in step (A) in a medium containing retinoic acid (RA) in a three-dimensional manner.

9. The method for producing according to claim 7, comprising: (A') three-dimensionally culturing adrenocortical-derived cells in a medium containing at least one of WNT and a substance that activates the WNT pathway, at least one of ACTH and a substance that activates the ACTH pathway, at least one of SHH and a substance that activates the SSH pathway, EGF, FGF10, FGF7, and FGF2; and (B') further three-dimensionally culturing the cells obtained in step (A') in a medium containing oncostatin M (OSM) and RA.

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