Method for producing milk-like products

Abstract

A method of producing mammalian breast milk exosomes, such as human breast milk exosomes, comprising generating lactating cells derived from mammalian breast epithelial cells, such as human breast epithelial cells, expressing mammalian milk-like products, such as human milk-like products, from the lactating cells, and purifying the exosomes from the mammalian milk-like products.

Description

Technical Field

[0001] This invention relates to an in vitro method for producing isolated mammalian breast milk exosomes, comprising: culturing and differentiating lactating cells derived from mammalian mammary epithelial cells, such as human mammary epithelial cells, and / or mammary gland-like organoids containing such lactating cells; expressing mammalian milk-like products, such as human milk-like products, from such lactating cells and / or mammary gland-like organoids; and purifying exosomes from the mammalian milk-like products. The invention also relates to isolated mammalian breast milk exosomes. The invention further relates to exosomes obtainable by this method. Background Technology

[0002] Mammal milk, especially human milk, is a complex fluid with multiple components, each contributing significantly to the health of the infant and even the mother. Growing evidence suggests that human breast milk is the most suitable source of nutrition for at least the first six months of life. Many components of human milk are either completely absent, present in very low amounts, or less active in cow's milk, and these components form the basis of infant formula. These include, for example, lactoferrin, growth factors, long-chain polyunsaturated fatty acids, or oligosaccharides. Human milk components have been used as the gold standard for developing current infant formula, and despite recent significant advancements in infant formula ingredients, it is impractical to assume that human milk or its components can be replicated using current manufacturing processes.

[0003] Currently, the only source of human milk is human donors (mothers who breastfeed). Donations for both non-commercial (human milk biobanks) and commercial purposes have been reported. However, breast milk donation is limited and subject to strict regulatory and safety controls, and sometimes ethical or religious constraints.

[0004] Stem cells found in mammalian milk, particularly human milk, are called human breast milk stem cells (hBSCs). hBSCs exhibit high adaptability to artificial insemination and can differentiate into multiple cell types in culture. More importantly, hBSCs can differentiate into the three cell lines required to form the lobular vesicle structures of human mammary glands (Hassiotou F. et al., Stem Cells, 2012). However, using hBSCs to produce human breast milk is neither practical nor sustainable because it requires human donors.

[0005] A cell line with stem cell function is currently known, and this technology is called induced pluripotent stem cells (iPSCs). A reliable two-step protocol for generating human breast-like organoids from human iPSCs (hiPSCs) has been proposed (Ying Qu et al., Stem Cell Report, Vol. 8, pp. 205-215, February 14, 2017).

[0006] Therefore, the object of this invention is to provide alternative and improved methods for producing mammalian milk and key components of mammalian milk, such as exosomes, using cultured cells without the use of early undifferentiated stem cells. Another object of this invention is to prepare customized mammalian milk-like products, such as human milk-like products, in cultured cells adapted to the specific needs of a recipient and / or to produce human milk bioactive substances such as exosomes to supplement existing milk-based infant nutrition solutions, again without the use of stem cells. A further object of this invention is to provide therapeutic uses of isolated human breast milk exosomes and compositions thereof. Summary of the Invention

[0007] The present invention solves the above-mentioned technical problems.

[0008] This article provides an isolated human breast milk exosome that does not contain one or more proteins selected from hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1).

[0009] This article also provides an isolated human breast milk exosome, wherein the exosome does not contain one or more proteins selected from C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0010] This article also provides an isolated human breast milk exosome in which hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) were not detected by liquid chromatography-mass spectrometry. This article also provides an isolated human breast milk exosome in which, under the same conditions as those detected in exosomes isolated from human breast milk by liquid chromatography-mass spectrometry, hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) were not detected by liquid chromatography-mass spectrometry.

[0011] This article also provides an isolated human breast milk exosome in which C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2) were not detected by liquid chromatography-mass spectrometry. This article also provides an isolated human breast milk exosome, wherein, under the same conditions as those under which C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2) were detected in exosomes isolated from human breast milk, these four substances were not detected by liquid chromatography-mass spectrometry.

[0012] This article also provides a population of exosomes, wherein the population comprises exosomes as described in any part of this article.

[0013] This document also provides a composition comprising exosomes or exosome populations as described in any part of this document.

[0014] The present invention also relates to exosomes as described in any part herein, exosome populations as described in any part herein, compositions as described in any part herein, or exosome products that can be obtained according to the methods described in any part herein for use in therapy.

[0015] This article also provides a method for treating a disease or disorder, comprising administering to a subject in need a therapeutically effective amount of exosomes, exosome populations, compositions, or exosome products that can be obtained according to the methods described in any part of this article.

[0016] This invention also relates to exosomes as described in any part of this document, exosome populations as described in any part of this document, compositions as described in any part of this document, or exosome products obtainable according to the methods described in any part of this document, for the prevention, treatment, or improvement of symptoms associated with inflammation. In some embodiments, the inflammation is intestinal inflammation. In some embodiments, the inflammation is bronchial inflammation. In some embodiments, the inflammation is respiratory tract inflammation.

[0017] This document also provides a method for preventing, treating, or improving symptoms associated with inflammation, the method comprising administering to a subject in need a therapeutically effective amount of exosomes, exosome populations, compositions, or exosome products obtainable according to methods described in any part of this document. In some embodiments, the inflammation is intestinal inflammation. In some embodiments, the inflammation is bronchial inflammation. In some embodiments, the inflammation is respiratory tract inflammation.

[0018] This document also provides a mammary exosome product, optionally a human breast milk exosome, that can be obtained according to the methods described in any part of this document.

[0019] Finally, this document provides a method for producing mammalian milk fortifiers, comprising performing the methods described in any part of this document. Detailed Implementation

[0020] definition

[0021] In the context of this invention, the term "in vitro" means that it is carried out or occurs elsewhere outside a test tube, petri dish, bioreactor, or biological organism.

[0022] In the context of this invention, the term "mammal" refers to an animal belonging to the mammalian species, such as humans, cattle, monkeys, camels, sheep, and goats.

[0023] In the context of this invention, the terms "lactating cells" or "mammary gland-like cells" refer to secretory epithelial cells that express the CK18 cell marker and are derived from mammalian mammary epithelial cells, particularly human mammary epithelial cells. As used herein, human mammary epithelial cells are commercially available and can be selected from any suitable cell line. In the context of this invention, suitable human mammary epithelial cell lines are, for example, non-tumorigenic cell lines such as MCF-10, or may be tumorigenic cell lines such as MCF-7.

[0024] In the context of this invention, the term "mammary gland-like organoid" or "mammary gland-like organoid" refers to a small and simplified mammary gland that grows in two or three dimensions (2D / 3D) and contains lactating cells as defined above.

[0025] In the context of this invention, the term "human milk-like product" refers to cell-cultured dairy products. It is an edible product expressed by lactating cells and / or mammary gland-like organoids generated according to the method of this invention.

[0026] The "human milk-like product" according to the invention may have the same components as the human milk of a well-nourished mother (e.g., in terms of bioactive substances, macronutrients, and micronutrients and their levels). This is referred to herein as a "standard human milk product". Alternatively, the "human milk-like product" according to the invention may have modified proportions and concentrations of components naturally present in the human breast milk of a well-nourished mother. This is referred to herein as a "non-standard milk-like product". The "human milk-like product" according to the invention may be modified such that it includes components not naturally present in the human breast milk of a well-nourished mother ("modified milk-like product"). Non-limiting examples of human milk-like products are selected from: supplements, fortifiers, human breast milk substitutes (or substitutes), and ingredients that are rich in only one and / or a portion of bioactive substances and macronutrients that are typically found in the breast milk of a well-nourished mother.

[0027] "Human milk-like products" can be used to replace the consumption of natural breast milk ("human milk substitutes"). Milk substitute products can be used as supplements ("human milk supplements") or as fortifiers ("human milk fortifiers") in combination with natural breast milk.

[0028] In one embodiment, the standard human milk-like product according to the invention comprises at least the macronutrients and micronutrients commonly found in the breast milk of well-nourished mothers. In one embodiment, the human milk-like product according to the invention comprises: proteins, peptides, lipids (including linoleic acid and α-linolenic acid), carbohydrates, vitamins (including vitamin A, vitamin D3, vitamin E, vitamin K, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, pantothenic acid, folic acid, vitamin C, and biotin), minerals (including iron, calcium, phosphorus, magnesium, sodium, chloride, potassium, manganese, iodine, selenium, copper, and zinc), choline, inositol, and L-carnitine. In one embodiment, the human milk-like product according to the invention further comprises at least one bioactive substance selected from: growth factors, cytokines, probiotics, extracellular vesicles (e.g., milk fat globules and / or exosomes), and bioactive substances derived from exosomes (e.g., miRNA) and secretory IgA. The standard human milk-like product according to the invention is not a product of naturally occurring human mammary gland secretion.

[0029] In another embodiment, the human milk-like product according to the invention can be adapted to the specific needs of the infant who will receive the product. The product may comprise only one and / or a portion of bioactive substances and macro and micronutrients typically found in the breast milk of well-nourished mothers. In such embodiments, the human breast milk-like product may also be referred to by the term "non-standard human milk-like product." In one embodiment, the non-standard human milk-like product according to the invention comprises one or more of the following nutrients or bioactive substances: proteins, peptides, lipids (including linoleic acid and α-linolenic acid), carbohydrates (including human milk oligosaccharides), vitamins (including vitamin A, vitamin D3, vitamin E, vitamin K, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, pantothenic acid, folic acid, vitamin C and biotin), minerals (including iron, calcium, phosphorus, magnesium, sodium, chloride, potassium, manganese, iodine, selenium, copper and zinc), choline, inositol, L-carnitine, growth factors, cytokines, probiotics, extracellular vesicles (e.g., milk fat globules and / or exosomes), bioactive substances derived from exosomes (e.g., miRNA), and secretory IgA.

[0030] In the context of this invention, the term "unmodified human milk-like product" refers to a human milk-like product expressed by lactating cells and / or mammary gland-like organoids generated by steps A) and B) of the method according to the invention, but not further processed by optional step C) of the method according to the invention. Unmodified human milk-like products can include both standard and non-standard human milk-like products. Non-limiting examples of non-standard human milk-like products are selected from: supplements, fortifiers, and ingredients containing only one and / or a portion of bioactive substances and macro and micronutrients typically found in the breast milk of well-nourished mothers.

[0031] In the context of this invention, the term "modified human milk-like product" refers to a human milk-like product expressed by lactating cells and / or mammary gland-like organoids generated by steps A) and B) of the method according to the invention and subjected to further processing by optional step C) of the method according to the invention.

[0032] Modified human milk-like products can include standard human milk-like products and non-standard human milk-like products.

[0033] In the context of this invention, the term "EB" refers to an embryoid.

[0034] In the context of this invention, the term "mEB" refers to "embryomorph cultured in MammoCult medium".

[0035] MammoCult medium is a serum-free medium containing basal medium, at least one proliferation supplement, heparin, and hydrocortisone.

[0036] In the context of this invention, the terms "embryomorph (EB)," "embryomorph cultured in MammoCult medium (mEB)," "mammary globule," and / or "globule" refer to a three-dimensional aggregate formed in suspension during step A) of the method of this invention.

[0037] In the context of this invention, the term "infant" refers to a child under 12 months of age, such as a child under 9 months of age, and particularly a child under 6 months of age.

[0038] In the context of this invention, the infant can be any full-term or premature infant. In one embodiment of the invention, the infant is selected from both premature and full-term infants.

[0039] The term "full-term baby" refers to a baby born at full term or 37 weeks of gestation or greater.

[0040] The term "premature baby" refers to a baby born at a gestational age of less than 37 weeks.

[0041] In the context of this invention, the term "birth weight" refers to the initial weight of a fetus or newborn obtained after birth.

[0042] In the context of this invention, the term "low birth weight" means a birth weight of less than 2500g (heaviest up to and including 2499g).

[0043] In the context of this invention, the term "very low birth weight" means a birth weight of less than 1500g (heaviest up to and including 1499g).

[0044] In the context of this invention, the term "very low birth weight" means a birth weight of less than 1000g (up to and including 999g).

[0045] The term "small for gestational age" refers to an infant whose birth weight is more than two standard deviations below the reference mean birth weight in a gestational growth chart, or whose birth weight is below the 10th percentile of population weight data obtained from infants of the same gestational age. The term "small for gestational age" includes infants whose birth size is smaller than average due to constitutive or genetic reasons or intrauterine growth restriction.

[0046] In the context of this invention, the term "toddler" or "walking child" refers to a child aged between 1 and 3 years.

[0047] As used in this article, the term "child" refers to a person who is older than three years but not older than twelve years.

[0048] As used in this article, the term "adult" refers to a person who is over twelve years of age.

[0049] As used herein, the term "infant formula" refers to a nutritional composition intended for use in infants, as defined in Codex Alimentarius, (Codex STAN 72-1981) and as defined in Codex Alimentarius, (Codex STAN 72-1981) as well as in Infant Specialities (incl. Food for Special Medical Purpose). It also refers to foods intended to provide specific nutritional purposes for infants in the first few months of life, which on their own meet the nutritional needs of such infants (in accordance with Article 2(c) of European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 concerning infant formula and Stage 2 formula). Infant formula encompasses Stage 1 infant formula and Stage 2 infant formula or follow-up formula. Typically, Stage 1 formula serves as a substitute for breast milk from birth, while subsequent Stage 2 formula serves as a substitute for breast milk from the sixth month of infancy.

[0050] "Growing milk" (or GUM) is offered starting from one year of age. It is typically a milk-based beverage designed to meet the specific nutritional needs of young children. These milk-based beverages are nutritional compositions intended to be fed to children aged 12 months to 2-3 years in combination with other foods.

[0051] In the context of this invention, the term "fortifier" refers to a composition containing one or more nutrients that have nutritional benefits for infants or young children.

[0052] The term "milk fortifier" refers to any composition used to fortify or supplement human breast milk, infant formula, growing milk, or human breast milk fortified with other nutrients. Therefore, the human milk fortifier of the present invention can be applied after being dissolved in human breast milk, infant formula, growing milk, or human breast milk fortified with other nutrients, or it can be applied as a standalone composition.

[0053] When applied as a standalone composition, the human milk fortifier of the present invention may also be identified as a “supplement.” In one embodiment, the milk fortifier of the present invention is a supplement.

[0054] The term "human milk fortifier" refers to any composition used to fortify or supplement human breast milk or human breast milk fortified with other nutrients. The "human milk fortifier" according to the invention is intended for use on premature infants, infants with very low birth weight (VLBW), or infants with extremely low birth weight (ELBW).

[0055] The emulsion fortifier according to the present invention can be in powder or liquid form.

[0056] Liquid emulsion fortifier compositions offer several specific advantages. For example, liquid formulations may be more convenient if they are to be associated with packaging that delivers a specific weight or volume of calibration droplets.

[0057] In addition, liquid formulations are easier to mix with the composition to be fortified, while powder formulations may clump in some cases.

[0058] In the context of this invention, the term "nutritional composition" means a composition that nourishes a subject. This nutritional composition is typically ingested orally or intravenously. It may contain a lipid or fat source, a carbohydrate source, and / or a protein source. In one specific embodiment, the nutritional composition is a ready-to-drink composition, such as a ready-to-drink formula food.

[0059] The EpiCult or EpiCultB medium mentioned in this article refers to a serum-free medium containing hydrocortisone, insulin, FGF10, and HGF.

[0060] As disclosed anywhere herein, a culture medium refers to a solid, semi-solid, or liquid containing essential nutrients and designed to support the growth and differentiation of microorganisms. MammoCult medium is an example of a culture medium that can be used in this invention.

[0061] Methods and uses

[0062] The present invention relates to a method for producing mammary gland cells using mammalian epithelial cells cultured under specific conditions described below, and a method for producing mammalian milk-like products in vitro using said mammary gland cells, wherein exosomes are purified from the mammalian milk-like products in order to isolate the exosomes.

[0063] It has been surprisingly demonstrated in this invention that mammary epithelial cells can be used as starting material in protocols for producing mammalian milk-like products. Using mammary epithelial cells as starting material means that complex differentiation protocols are not required to first produce mammary epithelial cells from, for example, early undifferentiated stem cells. Compared to stem cell protocols, this reduces the overall time of methods for producing mammalian milk-like products and offers cost-saving benefits.

[0064] Therefore, the present invention relates to a method for producing mammalian milk-like products, the method comprising:

[0065] A) Culture mammary epithelial cells in a culture medium to generate lactating mammary gland-like organoids; and

[0066] B) The mammalian milk-like product is secreted from the lactating cells.

[0067] mammalian milk product production

[0068] This invention relates to a method for producing mammalian milk-like products as defined herein, comprising any of steps A) and B) as defined herein, and an optional step C) as defined herein.

[0069] Step A — Generation of lactating cells and / or mammary gland-like organoids

[0070] According to the method of the present invention, mammary gland-like cells and / or organoid structures are generated in step A).

[0071] This includes enabling mammary epithelial cells to proliferate and mature to produce mammary-like organoids such as lactocytes.

[0072] In the method disclosed herein, mammary epithelial cells are used as the starting material; that is, the culture of epithelial cells is the first step of this method.

[0073] Mammary epithelial cells proliferate and mature by culturing them in a specific culture medium, such as complete MammoCult medium (StemCell Technologies). Preferably, complete MammoCult medium consists of basal medium, a proliferation supplement, heparin (typically 4 µg / mL), and hydrocortisone (typically 0.48 µg / mL). The medium is typically changed every three days. The mEBs (mammary globules) obtained in the above steps are then enriched with non-neuroectodermal cells.

[0074] In some embodiments, the proliferation and maturation phase occurs between day 0 and day 7, where day 0 is the time point at which mammary epithelial cells are first added to the culture medium. In some embodiments, the proliferation and maturation phase lasts for 7 days. In some embodiments, the proliferation and maturation phase lasts for no more than 7 days.

[0075] Following the proliferation and maturation phases, cells are induced to express milk proteins. In some embodiments, the induction period is between day 7 and day 14. In some embodiments, the induction period lasts for 7 days. In some embodiments, the induction period does not exceed 7 days.

[0076] In one embodiment of the invention, a method for producing a human milk-like product is provided, the method comprising: generating lactating cells from human mammary epithelial cells in step A), wherein such step A) includes:

[0077] i) Human mammary epithelial cells were cultured in a suitable culture medium (e.g., MammoCult medium) and lactating cells were generated after 7 days.

[0078] In another embodiment, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human mammary epithelial cells in step A), wherein such step A) includes:

[0079] i) Culture human mammary epithelial cells in a suitable culture medium (e.g., MammoCult medium) for at least 7 days under non-adherent conditions to generate lactating cells.

[0080] In one embodiment, the method according to the invention provides culture conditions according to step A), which are adapted to generate lactating cells derived from human mammary epithelial cells capable of secreting human milk-like products.

[0081] In a preferred embodiment, a method for producing a human milk substitute product is provided, the method comprising: generating lactating cells from human mammary epithelial cells in step A), wherein such step A) comprises: proliferating and maturing the mammary epithelial cells to differentiate them into mammary gland cells (e.g., lactating cells) in a suitable 3D culture system (e.g., 3D suspension conditions) as described anywhere herein. In some embodiments, this process lasts for at least 7 days. In some embodiments, it lasts for 7 days or less.

[0082] In another preferred embodiment, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human mammary epithelial cells in step A), wherein such step A) comprises:

[0083] i) Culture mammary epithelial cells in a suitable culture medium (e.g., MammoCult medium) for at least 7 days (day 0 to day 7) in a suitable 3D culture system (e.g., under 3D suspension conditions) to generate lactating cells.

[0084] In a particularly preferred embodiment of the invention, a method for producing a human milk-like product is provided, the method comprising: generating lactating cells from human mammary epithelial cells in step A), wherein:

[0085] i) Culture mammary epithelial cells in complete MammoCult medium (StemCell Technologies) for 7 days (day 0 to day 7). This complete MammoCult medium contains basal medium, proliferation supplement, and is supplemented with heparin (usually 4 µg / mL), hydrocortisone (usually 0.48 µg / mL), and

[0086] ii) Milk protein expression was induced by incubating cells in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days (day 7 to day 14).

[0087] Step ii) preferably leads to the differentiation of milk protein-expressing cells, especially lactating cells and / or mammary gland-like organoids.

[0088] In another particularly preferred embodiment of the invention, a method for producing a human milk-like product is provided, the method comprising: generating lactating cells from human mammary epithelial cells in step A), wherein:

[0089] i) Culture mammary epithelial cells in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, and heparin as described anywhere in this document for 7 days (day 0 to day 7), and

[0090] ii) Milk protein expression was induced by incubating cells in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days (day 7 to day 14).

[0091] Step ii) preferably leads to the differentiation of milk protein-expressing cells, especially lactating cells and / or mammary gland-like organoids.

[0092] In one embodiment, step ii) as defined above for a particularly preferred embodiment preferably results in the formation / differentiation of at least mammary cells, luminal cells, and basal cells. In this context, mammary cells preferably express one or more markers, preferably all selected from the following: β-casein, milk proteins, and hormone receptors. Furthermore, luminal cells preferably express one or more markers, preferably all selected from the following: EpCAM, MUC1, CD49F, GATA3, CK8, and CK18. Furthermore, basal cells preferably express one or more markers selected from the following: CK14, α-smooth muscle actin, and P63.

[0093] In another embodiment, following step ii) as defined above for a particularly preferred embodiment, a mammary gland-like organoid expressing one or more of the following markers can be obtained: β-casein, milk proteins, and hormone receptors; luminal cells expressing one or more of the following markers: EpCAM, MUC1, CD49F, GATA3, CK8, CK18; and basal cells expressing one or more of the following markers: CK14, α-smooth muscle actin, and P63.

[0094] In one embodiment of the present invention, the above-described method for producing human milk-like products is provided.

[0095] In one embodiment of (step A), the delivery of nutrients and biomimetic stimuli is controlled to influence cell growth, differentiation, and tissue formation. In one embodiment of (step A), such control is carried out in a bioreactor.

[0096] In some embodiments, the mammary gland-like organoids or lactating cells derived from step A have high expression of mammary gland-specific markers. In some embodiments, the mammary gland-like organoids or lactating cells derived from step A express keratin 18 (KRT-18). In some embodiments, the mammary gland-like organoids or lactating cells derived from step A express estrogen receptor (ER). In some embodiments, the mammary gland-like organoids or lactating cells derived from step A express more than 59% KRT-18 and ER as an estrogen receptor-positive luminal mammary gland population.

[0097] In some embodiments, mammary gland-like organoids or lactating cells derived from step A exhibit high expression of key lactogenic bioactive substances. In some embodiments, mammary gland-like organoids or lactating cells derived from step A exhibit increased expression of key lactogenic bioactive substances. In some embodiments, compared to pre-induction (e.g., day 7), mammary gland-like organoids or lactating cells derived from step A exhibit increased expression of key lactogenic bioactive substances after induction (e.g., day 14). In some embodiments, compared to pre-induction, mammary gland-like organoids or lactating cells derived from step A exhibit increased mRNA expression of lactoferrin (LTF) after induction. In some embodiments, compared to pre-induction, mammary gland-like organoids or lactating cells derived from step A exhibit increased mRNA expression of milk fat globule-EGF factor 8 (MFGE8) after induction.

[0098] It should be understood that any methods or method steps disclosed herein can be performed in 3D suspension cultures, rather than using a membrane matrix as a support. Therefore, in some embodiments, cells are maintained in suspension cultures throughout the differentiation process.

[0099] Step B—Expression of human breast milk-like product

[0100] In one embodiment of the invention, the method includes expressing a human milk-like product from mammary gland-like organoids derived from human mammary epithelial cells, preferably prepared according to step A). ​​Preferably, the human milk-like product is expressed after inducing expression of human milk-like products from such lactating cells and / or mammary gland-like organoids.

[0101] In one implementation, lactating cells are induced to produce lactation by applying a specific culture medium (e.g., EpiCultB) supplemented with lactation-inducing factors (e.g., prolactin, hydrocortisone, and insulin).

[0102] Specifically, the human milk-like product obtained from mammary gland-like organoids derived from human mammary epithelial cells preferably prepared according to step A) contains bioactive substances from human milk, selected from or consisting of the group consisting of proteins, lipids, or oligosaccharides, preferably human milk oligosaccharides, etc. Specifically, the inventors have sought to maintain consistency with the particularly preferred schemes performed according to steps A) i) to iv) as described above, especially with respect to oligosaccharides (including lactose and some HMOs), lipids (including four fatty acids), proteins (seven detected, including casein), and miRNAs (75 detected, including 11 commonly detected in HBM).

[0103] In one embodiment, the human milk-like product obtained from a mammary gland-like organoid derived from human mammary epithelial cells preferably prepared according to step A) contains bioactive substances from human milk selected from or consisting of the group consisting of oligosaccharides, lipids, proteins, exosomes, and miRNAs. In one embodiment, the human milk-like product obtained from a mammary gland-like organoid derived from human induced pluripotent stem cells (hiPSCs) (preferably prepared according to step A) comprises exosomes.

[0104] In another embodiment, the human milk-like product obtained from mammary gland-like organoids derived preferably from human mammary epithelial cells prepared according to step A) contains bioactive substances from human milk, which are selected from the group consisting of or comprise the following: lactose, 6'SL, C-4:0 fatty acids, C-8:0 fatty acids, C-10:0 fatty acids, C-14:0 fatty acids, C-15:0 fatty acids, C-16:0 fatty acids, C-16:1n7 fatty acids, C-17:0 fatty acids, C-18:0 fatty acids, C-18:1n9 fatty acids, C-18:1 fatty acids, C-18:2n6 fatty acids, C-20:0 fatty acids, C-20:1n9 fatty acids, C-18:3n3 fatty acids, C-22:0 fatty acids, lactoferrin, albumin, prolactin, α-lactalbumin ... S1-casein, hemoglobin β subunit, hemoglobin α subunit, α-lactalbumin, α-2-macroglobulin, β-casein, bile salt activated lipase, κ-casein, lactoglucosin, CD14, fatty acid synthase, IgA, pIgR, serum albumin, xanthine dehydrogenase, exosomes, miR-21-5p, miR-181a-5p, miR-30d-5p, miR-30b-5p, miR-22-3p, miR-146b-3p, miR-30c-5p, miR-30a-5p, miR-30e-5p, and miR-148b-3p.

[0105] In one embodiment of the present invention, the human milk-like product obtained from mammary gland-like organoids derived from human mammary epithelial cells is a standard human milk product. In another embodiment of the present invention, the human milk-like product obtained from mammary gland-like organoids derived from human mammary epithelial cells is a non-standard human milk product.

[0106] Step C—Further processing to produce a modified human breast milk-like product.

[0107] In an optional embodiment of the invention, the method described herein includes an additional step C), which is performed on a human milk-like product that can be obtained from step B), and includes performing additional processing on such product to provide a modified human milk-like product.

[0108] In one particular embodiment, the additional processing step C) performed on the human breast milk-like product of the present invention may be selected from: purification step, separation process, extraction process, fractionation step, enrichment process, enzyme treatment, addition of additional components (e.g., components that cannot be expressed by glandular organoids of human mammary glands (such as, for example, immunoglobulins, probiotics and / or minerals) or combinations thereof).

[0109] Specifically, in some embodiments, the mammalian milk-like products described in any part of this document are extracted, separated, and purified as part of the methods described in any part of this document. Therefore, the present invention provides an isolated mammalian milk-like product, such as an isolated human milk-like product.

[0110] In some embodiments, the isolated mammalian milk-like products are formulated into compositions. In some embodiments, the isolated mammalian milk-like products are formulated into nutritional compositions. In some embodiments, the isolated human milk-like products are formulated into pharmaceutical compositions.

[0111] In some embodiments, the isolated mammalian milk-like products are formulated as compositions. In some embodiments, the isolated mammalian milk-like products are formulated as nutritional compositions. In some embodiments, the isolated mammalian milk-like products are formulated as pharmaceutical compositions.

[0112] In some embodiments, specific mammalian milk-like bioactive substances are extracted, isolated, and purified from mammalian milk-like products, selected from or consisting of the group consisting of oligosaccharides, lipids, proteins, exosomes, and miRNAs. In particular, exosomes are extracted, isolated, and purified from mammalian milk-like products. Therefore, the present invention provides isolated mammalian milk-like bioactive substances selected from or consisting of the group consisting of oligosaccharides, lipids, proteins, exosomes, and miRNAs. In particular, the present invention provides isolated exosomes extracted, isolated, and purified from mammalian milk-like products, such as isolated human exosomes extracted, isolated, and purified from human milk-like products.

[0113] In some embodiments, the isolated bioactive substances from mammalian milk products are formulated into compositions. In some embodiments, the isolated bioactive substances from mammalian milk products are formulated into nutritional compositions. In some embodiments, the isolated bioactive substances from human milk products are formulated into pharmaceutical compositions.

[0114] In some embodiments, the isolated bioactive substances from mammalian milk products are formulated into compositions. In some embodiments, the isolated bioactive substances from mammalian milk products are formulated into nutritional compositions. In some embodiments, the isolated bioactive substances from mammalian milk products are formulated into pharmaceutical compositions.

[0115] In some embodiments, isolated mammalian milk-like exosomes are formulated as compositions. In some embodiments, isolated mammalian milk-like exosomes are formulated as nutritional compositions. In some embodiments, isolated human milk-like exosomes are formulated as pharmaceutical compositions.

[0116] In some embodiments, the isolated mammalian milk-like product exosomes are formulated as compositions. In some embodiments, the isolated mammalian milk-like product exosomes are formulated as nutritional compositions. In some embodiments, the isolated mammalian milk-like product exosomes are formulated as pharmaceutical compositions.

[0117] In some embodiments, step C) includes purifying exosomes from the human milk-like product. In some embodiments, step C) includes purifying exosomes from the human milk-like product to remove impurities, such as by chromatography, filtration, or ultracentrifugation, to separate the exosomes. In some embodiments, step C) includes purifying exosomes from the human milk-like product by chromatography to separate the exosomes. In some embodiments, step C) includes purifying exosomes from the human milk-like product by filtration to separate the exosomes. In some embodiments, step C) includes purifying exosomes from the human milk-like product by ultracentrifugation to separate the exosomes.

[0118] In some implementations, the chromatography method is column-based chromatography, such as size inclusion or size exclusion chromatography.

[0119] It should be understood that the products obtained from the purification process will differ in composition from those obtained from the unpurified process. In particular, the surface composition of the products will differ because components (such as proteins) attached to the surface of the product molecules may be removed during the purification steps. For example, proteins present on the surface of exosomes may be removed.

[0120] In some implementations, step C) further includes sterilizing the isolated exosomes.

[0121] In some implementations, step C) further includes storing the isolated exosomes at a temperature below freezing (optionally -80°C).

[0122] In some embodiments, step C) includes formulating the isolated exosomes into a powder form (e.g., by freeze-drying or spray-drying) or a liquid form.

[0123] In some implementations, the method also includes formulating isolated exosomes together with supplemental nutritional ingredients.

[0124] In some implementations, the method also includes aliquoting the isolated exosomes into containers for consumption.

[0125] In some embodiments, the isolated exosomes express a four-span membrane protein. In some embodiments, the isolated exosomes express CD9, CD63, and / or CD81.

[0126] Human milk-like products

[0127] "Standard" human breast milk-like products

[0128] In one embodiment of the invention, the human breast milk-like product is a "standard" human breast milk-like product, that is, it contains the same components as the human breast milk of a well-nourished mother.

[0129] According to scientific research literature, the beneficial effects of breastfeeding are well known, and the possibility of using human breast milk-like products brings many of the same well-known health benefits.

[0130] In such implementations, human breast milk-like products can be used as a substitute for breastfeeding when actual breastfeeding is not possible.

[0131] In such implementations, human breast milk-like products are intended to be used, for example, to support women who produce less milk or who have stopped lactating 6 months after childbirth to extend the duration of breastfeeding.

[0132] Similarly, human breast milk-like products are intended for use, for example, to allow breastfeeding to be achieved when a mother is unable to actually breastfeed due to illness.

[0133] In another implementation, the human breast milk-like product is intended for use in cases where breast milk cannot begin to be produced naturally, for example, when the infant is adopted.

[0134] In one embodiment, the human milk-like product according to the invention is not a product of naturally occurring human breast milk secretion.

[0135] In one implementation, human breast milk-like products are used to provide optimal nutrition for infants.

[0136] In one implementation, human breast milk-like products are used to promote the healthy growth of infants.

[0137] In one implementation, human breast milk-like products are used to prevent infant infections and obesity, as well as to promote infant immune development.

[0138] In one implementation, the human breast milk-like product is an unmodified human breast milk-like product.

[0139] In another embodiment, the human breast milk-like product is a modified human breast milk-like product.

[0140] In one embodiment, the human milk-like product according to the invention comprises: proteins, lipids, carbohydrates, vitamins, and minerals.

[0141] In another embodiment, the human milk-like product according to the present invention comprises: proteins, lipids, carbohydrates, vitamins, minerals, and bioactive substances.

[0142] In one embodiment, the human milk-like product according to the invention comprises: proteins, lipids (including linoleic acid and alpha-linolenic acid), carbohydrates, vitamins (including vitamin A, vitamin D3, vitamin E, vitamin K, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, pantothenic acid, folic acid, vitamin C and biotin), minerals (including iron, calcium, phosphorus, magnesium, sodium, chloride, potassium, manganese, iodine, selenium, copper and zinc), choline, inositol and L-carnitine.

[0143] In another embodiment, the human milk-like product according to the invention further comprises at least one bioactive substance selected from: growth factors, cytokines, probiotics, extracellular vesicles (e.g., milk fat globules and / or exosomes) and bioactive substances derived from exosomes (e.g., miRNAs) and secretory IgA.

[0144] Such human breast milk-like products can be prepared by the method according to the invention, for example by step C) including the addition of growth factors, cytokines, probiotics, extracellular vesicles (e.g., milk fat globules and / or exosomes), bioactive substances from exosomes (e.g., miRNAs), and secretory IgA.

[0145] In one implementation, the human breast milk-like product contains probiotics.

[0146] Such human breast milk-like products can be prepared according to the method of the present invention, for example by step C) including adding probiotics (e.g., Bifidobacterium lactis, Bifidobacterium infantis, and Lactobacillus rhamnosus) available from several commercially available sources.

[0147] In such implementations, human breast milk-like products can be used to optimize gastrointestinal function and / or promote immunity.

[0148] In one implementation, the human breast milk-like product contains secretory IgA and probiotics.

[0149] Such human breast milk-like products can be prepared according to the method of the invention, for example by including step C): adding a combination of probiotics and secretory IgA, which can be prepared as described, for example, in patent applications WO2009 / 156301 and WO2009 / 156367, which are hereby incorporated by reference. In such embodiments, the human breast milk-like products can be used to prevent immunoglobulin deficiency and / or to prevent recurrent infections in infants and young children.

[0150] Non-standard human breast milk-like products

[0151] In one embodiment of the invention, the human milk-like product may have altered proportions and concentrations of components naturally present in the breast milk of a well-nourished mother. This is referred to herein as a "non-standard milk-like product".

[0152] In one embodiment, the human milk-like product according to the invention may be selected from: milk fortifiers, supplements, and / or human breast milk substitutes adapted for specific purposes.

[0153] Human milk fortifiers and human milk bioactive supplements

[0154] In one embodiment, the method of the present invention provides a human breast milk-like product that can be used to fortify natural human breast milk obtained from lactating mothers or to fortify infant formula.

[0155] In another embodiment, the method of the present invention provides a human breast milk-like product that can be used as a supplement for infants or young children in need.

[0156] In such implementations, human breast milk-like products can be used to provide healthy growth and / or reduce the risk of developing diseases typically associated with specific conditions in infants or young children (such as, for example, asthma, allergies, cognitive changes) and / or promote catching up with growth rates, developing immunity, and preventing infections.

[0157] It is worth noting that, in light of the facts presented by the method according to the invention, human-derived ingredients (especially bioactive ingredients) in such fortifiers or supplements should maintain the full or enhanced functionality of such ingredients.

[0158] Preferably, the human breast milk-like product is intended for use as a fortifier. Such a human breast milk-like product is intended for use as a fortifier and can be prepared according to the method of the invention, for example, by step C) comprising isolating and / or enriching (certain) bioactive substances from the human breast milk-like product obtainable from step B). Such isolation steps can be performed by classical fractionation, enrichment, and / or purification of the unmodified human breast milk-like product obtainable from step B).

[0159] Human breast milk-like products intended for use as supplements may contain one or more bioactive substances selected from: human milk oligosaccharides (e.g., 2FL, 3FL, LNT, LnNT, DiFl, 6SL, and / or 3SL), lipids, growth factors (e.g., epidermal growth factor (EGF), heparin-bound epidermal growth factor), cytokines (e.g., transforming growth factor β2 (TGFβ-2)), and IL-1, IL-2, IL-6, IL-10, IL-18, interferon-γ (INF-γ), TNF-α, extracellular vesicles (e.g., milk fat globules and / or exosomes), exosomes including microRNAs, and antimicrobial / protective bioactive substances (e.g., IgA, lactoferrin, lysozyme, lactoglucosin). Such human breast milk-like products intended for use as supplements may be prepared according to the method of the invention, for example, by step C) comprising isolating these bioactive substances from an unmodified human breast milk-like product that can be obtained by step B). Such separation steps can be performed by classical fractionation, enrichment, and / or purification of the unmodified human breast milk-like product that can be obtained from step B).

[0160] In one embodiment, the human breast milk-like product is a supplement or milk fortifier containing fucosylated human milk oligosaccharides, such as 2FL and / or 3FL. Such supplements or milk fortifiers are intended to enhance the characteristics of human breast milk from women who do not secrete fucosylated oligosaccharides due to inactivity of the FUT2 gene.

[0161] Such human breast milk-like products intended for use as fortifiers or supplements can be prepared according to the method of the invention, for example by step C) comprising isolating and / or enriching fucosylated oligosaccharides (e.g., 2FL and / or 3FL) from unmodified human breast milk-like products that can be obtained by step B).

[0162] In such implementations, human breast milk-like products can be used to optimize gastrointestinal function and / or promote immunity.

[0163] Human breast milk-like products for infants with genetic diseases

[0164] In one embodiment, the human breast milk-like product according to the invention can be adapted to address the specific needs of infants with congenital genetic diseases.

[0165] Galactosemia

[0166] In such implementations, human breast milk-like products can be adapted to the needs of infants with galactosemia, a rare genetic disorder that affects an infant's ability to metabolize galactose.

[0167] In such embodiments, the human breast milk-like product should be lactose-free and / or contain lactose-containing sugars. In such embodiments, the human breast milk-like product can be used to promote the healthy growth of infants affected by galactosemia.

[0168] In one embodiment, according to the method of the invention, a human breast milk-like product free of lactose and / or containing lactose sugars can be obtained by step C), which includes enzymatic treatment (lactase treatment) or membrane fractionation and ultrafiltration of the unmodified human breast milk-like product that can be obtained by step B).

[0169] phenylketonuria

[0170] In such implementations, human breast milk-like products can be adapted to the needs of infants with phenylketonuria (PKU). PKU occurs due to the absence or inability of phenylalanine hydroxylase, the enzyme that converts phenylalanine to tyrosine. If left untreated, this condition can lead to brain toxicity and severe intellectual disability.

[0171] In such implementations, human breast milk-like products should contain no or have reduced levels of phenylalanine.

[0172] In such implementations, human breast milk-like products can be used to promote the healthy growth of infants affected by PKU.

[0173] In one implementation, the human breast milk-like product reduces phenylalanine in such a way that the phenylalanine level is kept below 20 mg / kg relative to the body weight of the subject receiving phenylalanine.

[0174] In one embodiment, according to the method of the invention, a human breast milk-like product with reduced or no phenylalanine can be obtained by step C), which includes enzymatic treatment (protein hydrolysis) or filtration of the unmodified human breast milk-like product that can be obtained by step B).

[0175] In one embodiment, according to the method of the invention, a human breast milk product with reduced phenylalanine content can be obtained by step C), which includes enzymatic treatment (protein hydrolysis) or filtration of the unmodified human breast milk-like product that can be obtained from step B).

[0176] In another embodiment, according to the method of the invention, a human breast milk-like product with reduced phenylalanine can be obtained by providing a culture medium (such as, for example, a glycomacropeptide (GMP) culture medium from whey) in step B) that provides a limited amount or no phenylalanine.

[0177] Components of human milk-like products

[0178] As previously described herein, the human milk-like product according to the present invention comprises at least one bioactive substance. One such bioactive substance is an exosome.

[0179] exosomes

[0180] Human breast milk exosomes are tiny microvesicles involved in infant development, such as in immunity, metabolism, and growth. They are believed to play an important role in treatment and disease prevention.

[0181] Exosomes consist of a lipid bilayer membrane that encapsulates a molecular combination including proteins, lipids, messenger RNA (mRNA), and microRNA (miRNA). The types and concentrations of these molecules determine the fingerprint and function of exosomes.

[0182] This invention provides isolated human breast milk exosomes. The exosomes of this invention have the same lipid bilayer vesicle structure and function as naturally derived human breast milk exosomes.

[0183] The exosomes of this invention are not produced from naturally occurring human breast milk.

[0184] In some embodiments, the present invention provides exosomes isolated from human milk-like products described in any part thereof, wherein the human milk-like products are produced by any of the methods described herein.

[0185] Therefore, for example, the present invention relates to an in vitro method for producing exosomes, the method comprising:

[0186] include:

[0187] A) Culture mammary epithelial cells in a culture medium to generate lactating mammary gland-like organoids; and

[0188] B) The lactating cells secrete mammalian milk-like products, and

[0189] C) Purify the exosomes from the mammalian milk product to remove impurities, optionally by chromatography, filtration, or ultracentrifugation, to separate the exosomes.

[0190] Optionally, step A) can be performed under 3D suspension conditions.

[0191] The exosomes of this invention differ in composition from those derived from natural human breast milk.

[0192] In some embodiments, the exosomes of the present invention differ in proteomic profile from those derived from natural human breast milk. It should be understood that proteomic profile refers to the types and / or concentrations of proteins present in the exosomes.

[0193] In some embodiments, the exosomes of the present invention are substantially identical to exosomes derived from natural human breast milk in one or more of the following characteristics: size, miRNA profile, and / or lipid profile. In some embodiments, the size, miRNA profile, and lipid profile of the exosomes of the present invention are substantially identical to those of exosomes derived from natural human breast milk.

[0194] In some implementations, the difference in proteomic profile is the only difference between the exosomes of the present invention and exosomes derived from natural human breast milk.

[0195] This invention also relates to exosome populations as described in any part herein. In some embodiments, the exosomes in the population are the same or different in terms of their structure, function, and / or composition. In some embodiments, the exosomes in the population are identical. In some embodiments, the exosomes in the population are different in composition.

[0196] In some embodiments, exosomes or exosome populations as described herein are formulated in powder form (e.g., by freeze-drying or spray-drying) or liquid form. In some embodiments, exosomes or exosome populations as described herein are frozen, optionally at -80°C. In some embodiments, exosomes or exosome populations as described herein are sterilized.

[0197] In some implementations, exosomes or exosome groups, as described herein, are packaged in containers for consumption.

[0198] In some embodiments, the present invention relates to a composition comprising exosomes or exosome populations as described in any part of this document. In some embodiments, the present invention relates to a nutritional composition comprising exosomes or exosome populations as described in any part of this document.

[0199] In some embodiments, the present invention provides a composition comprising exosomes or exosome populations as described in any part herein. In some embodiments, the composition is a nutritional composition. In some embodiments, the composition is a pharmaceutical composition.

[0200] In some embodiments, the composition contains supplemental nutrients.

[0201] In some embodiments, the present invention provides a pharmaceutical composition comprising exosomes or exosome groups as described in any part herein, and pharmaceutically acceptable excipients.

[0202] As used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeias for animal (more specifically for human) use.

[0203] In some embodiments, the compositions (e.g., nutritional or pharmaceutical compositions) described in any part of this document comprise exosomes isolated from human milk-like products described in any part of this document, wherein the human milk-like products are produced by any of the methods described herein.

[0204] Therefore, for example, the present invention relates to a composition comprising exosomes (e.g., a nutritional or pharmaceutical composition), wherein the exosomes are produced according to an in vitro method comprising:

[0205] include:

[0206] A) Culture mammary epithelial cells in a culture medium to generate lactating mammary gland-like organoids; and

[0207] B) The lactating cells secrete mammalian milk-like products, and

[0208] C) Purify the exosomes from the mammalian milk product to remove impurities, optionally by chromatography, filtration, or ultracentrifugation, to separate the exosomes.

[0209] Optionally, step A) can be performed under 3D suspension conditions.

[0210] In some embodiments, the compositions described in any part of this document are formulated as powder (e.g., by freeze-drying or spray drying) or as liquid. In some embodiments, the compositions described in any part of this document are frozen, optionally at -80°C.

[0211] In some embodiments, the compositions described in any part of this document are packaged into containers for consumption.

[0212] Exosomal proteomics

[0213] Through proteomics analysis, approximately 2,000 proteins have been identified in human breast milk exosomes in the literature.

[0214] The inventors of this invention performed proteomic analysis on the exosomes of this invention and unexpectedly demonstrated that these exosomes possess a proteome similar to, but not identical to, that of exosomes derived from natural human breast milk.

[0215] In particular, it has been found that the exosomes of the present invention lack one or more proteins present in exosomes derived from natural human breast milk. In some embodiments, these proteins include, but are not limited to, hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1). In some embodiments, these proteins include, but are not limited to, C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0216] In some embodiments, hypoxia-upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) were not detected in the human breast milk exosomes isolated by the present invention by liquid chromatography-mass spectrometry. In some embodiments, hypoxia-upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) were detected in exosomes isolated from human breast milk by liquid chromatography-mass spectrometry under the same conditions.

[0217] In some embodiments, C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2) were not detected in the human breast milk exosomes isolated by liquid chromatography-mass spectrometry according to the present invention. In some embodiments, under the same conditions, C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2) were detected in the exosomes isolated from human breast milk by liquid chromatography-mass spectrometry.

[0218] In this invention, exosome protein profiles are detected by liquid chromatography-mass spectrometry (LC-MS). The sensitivity of LC / MS analysis is at the femtomolar (fmol) level.

[0219] Exemplary, and not limiting, suitable mass spectrometers include the Orbitrap Fusion. ™ Lumos ™ Tribrid ™ Mass spectrometer. Exemplary, and not limiting, suitable HPLC systems include Vanquish. ™ The Neo UHPLC system, 300 nL / min, and exemplary, non-limiting, suitable HPLC columns include Acclaim ™ PepMap ™ RSLC C18 2µm 50cm×75μm. The total HPLC gradient time is, for example, about 100 min to about 200 min, preferably about 180 min. In some embodiments, the total HPLC gradient time is 180 min.

[0220] In another implementation, the mass spectrometer is an Orbitrap Fusion. ™ Lumos ™ Tribrid ™ The mass spectrometer and HPLC system is Vanquish. ™ Neo UHPLC system, 300 nL / min, and the HPLC column is Acclaim. ™ PepMap ™ RSLC C18 2µm 50cm × 75μm. In some implementations, the mass spectrometer is an Orbitrap Fusion. ™Lumos ™ Tribrid ™ The mass spectrometer and HPLC system is Vanquish. ™ Neo UHPLC system, 300 nL / min, and the HPLC column is Acclaim. ™ PepMap ™ RSLC C18 2µm 50cm×75μm, the total HPLC gradient time used was 180min.

[0221] The amount of protein used to prepare samples for proteomics LC / MS analysis is from about 15 µg to about 50 µg, preferably from about 20 µg to about 35 µg, and more preferably from about 25 µg. The amount of peptide injected per LC MS / MS analysis (i.e., column loading) is from about 0.5 µg to about 5 µg, preferably from about 1 µg to about 5 µg, and more preferably from about 1 µg to about 2.5 µg. In some embodiments, the amount of protein used to prepare samples for proteomics LC / MS analysis is about 1 µg. In other embodiments, the amount of protein used to prepare samples for proteomics LC / MS analysis is about 2.5 µg.

[0222] In some implementations, the mass spectrometer is an Orbitrap Fusion. ™ Lumos ™ Tribrid ™ The mass spectrometer and HPLC system is Vanquish. ™ Neo UHPLC system, 300 nL / min, HPLC column is Acclaim ™ PepMap ™ RSLC C182µm 50cm×75μm, and the amount of protein used to prepare the sample for proteomics LC / MS analysis is about 1µg, optionally, the total HPLC gradient time is 180min. In another embodiment, the mass spectrometer is an Orbitrap Fusion. ™ Lumos ™ Tribrid ™ The mass spectrometer and HPLC system is Vanquish. ™ Neo UHPLC system, 300 nL / min, HPLC column is Acclaim ™ PepMap ™ The RSLC C18 2µm 50cm×75μm was used, and the amount of protein used to prepare the sample for proteomics LC / MS analysis was about 2.5µg. Optionally, the total HPLC gradient time used was 180min.

[0223] Protein identification is performed using software such as, for example, Mascot v2.8.2 and Scaffold v5.0.0. Databases used for protein identification include, for example, the SwissProt Human Database (20221118, 20607 entries). The standard for protein identification is a peptide false detection rate (FDR) of 1% (minimum 1 or 2 peptides). FDR involves the probability of correctly identifying a peptide / protein. Therefore, a 1% FDR means that out of a list of 100 identified proteins, there is a possibility that 1 protein was incorrectly identified.

[0224] Exemplary LC / MS detection conditions are provided in the table below:

[0225]

[0226] In some embodiments, one or more of hypoxia-upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) were not detected in the exosomes of the present invention by LC / MS analysis as described herein.

[0227] In some embodiments, by LC / MS analysis as described herein, one or more of the following substances were not detected in the exosomes of the present invention: C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0228] In some embodiments, hypoxia upregulated protein 1 (HYOU1) was not detected in the exosomes of the present invention by LC / MS analysis as described herein.

[0229] In some embodiments, apolipoprotein A-IV (APOA4) was not detected in the exosomes of the present invention by LC / MS analysis as described herein.

[0230] In some embodiments, procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) was not detected in the exosomes of the present invention by LC / MS analysis as described herein.

[0231] As used herein, the term "not detected" includes the absence of a protein or peptide in a sample, or the presence of a protein or peptide in such low amounts that it cannot be detected by LC / MS analysis, for example, at a concentration below fmol. In some embodiments, reference to "not detected proteins and peptides" means that, according to any of the embodiments described above for detection by LC / MS, the protein and peptide sequences were not detected by LC / MS analysis.

[0232] However, importantly, the exosomes of the present invention contain proteins known in the literature to be present in exosomes derived from natural human breast milk and essential for infant health and development. These proteins include lactoferrin (LTF), annexin A2 (ANXA2), and lactoglossin (MFGE8). In some embodiments, these proteins include lactoferrin (LTF), annexin A2 (ANXA2), lactoglossin (MFGE8), MUC1, and the tetraspan membrane proteins CD9, CD81, and CD63.

[0233] Therefore, in some embodiments, the present invention provides a human breast milk exosome, wherein the exosome does not contain one or more proteins selected from C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0234] In some implementations, the exosome does not contain C4a anaphylatoxin (CFA).

[0235] In some implementations, the exosome does not contain hypoxia upregulated protein 1 (HYOU1).

[0236] In some implementations, the exosome does not contain apolipoprotein A-IV (APOA4).

[0237] In some implementations, the exosome does not contain procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1).

[0238] In some implementations, the exosome does not contain cytoplasmic threonine-tRNA ligase 1 (TARS1).

[0239] In some implementations, the exosome does not contain a cytoplasmic nonspecific dipeptidase (CNDP2).

[0240] In some embodiments, the exosome does not contain one or more of the following: hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1).

[0241] In some embodiments, the exosome does not contain the following proteins: C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0242] In some embodiments, the exosome contains one or more proteins listed in Table 1. In some embodiments, the exosome contains all the proteins listed in Table 1.

[0243] Table 1

[0244]

[0245] In some embodiments, the exosome comprises lactoferrin (LTF), annexin A2 (ANXA2), and / or lactoglossin (MFGE8). In some embodiments, the exosome comprises lactoferrin (LTF), annexin A2 (ANXA2), and lactoglossin (MFGE8). In some embodiments, the exosome comprises lactoferrin (LTF), annexin A2 (ANXA2), lactoglossin (MFGE8), MUC1, and the tetraspan membrane proteins CD9, CD81, and CD63.

[0246] In some embodiments, the exosome contains one or more of the proteins described in any part of this document at a concentration substantially the same as that of the same one or more proteins found in exosomes derived from natural human breast milk.

[0247] In some embodiments, the exosome contains one or more of the proteins described in any part of this document at a concentration different from the concentration of the same one or more proteins in exosomes derived from natural human breast milk. In some embodiments, the one or more proteins can be present at a higher concentration than the concentration of the one or more proteins in exosomes derived from natural human breast milk. In some embodiments, the one or more proteins can be present at a lower concentration than the concentration of the one or more proteins in exosomes derived from natural human breast milk.

[0248] As described herein, in some embodiments, the present invention relates to a composition comprising exosomes or exosome populations as described in any part thereof. In other embodiments, the present invention relates to a composition comprising: i) exosomes or exosome populations as described in any part thereof, and ii) one or more proteins not present in the exosomes of the present invention, but present in exosomes derived from natural human breast milk. The proteins may be selected from, but are not limited to, one or more of the following proteins: C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0249] In some embodiments, the composition is a nutritional composition. Therefore, in some embodiments, the present invention relates to a nutritional composition comprising: i) exosomes or exosome populations as described in any part herein, and ii) one or more proteins not present in the exosomes of the present invention, but present in exosomes derived from natural human breast milk. The proteins may be selected from, but are not limited to, one or more of the following proteins: hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1). In another embodiment, the protein may be selected from, but is not limited to, one or more of the following proteins: C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0250] In some embodiments, the composition is a pharmaceutical composition. Therefore, in some embodiments, the present invention relates to a pharmaceutical composition comprising: i) exosomes or exosome populations as described in any part herein, ii) a pharmaceutically acceptable excipient, and iii) one or more proteins not present in the exosomes of the present invention, but present in exosomes derived from natural human breast milk. Hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1). In another embodiment, the protein may be selected from, but is not limited to, one or more of the following proteins: C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2).

[0251] Exosomal miRNA profile

[0252] The inventors of this invention performed miRNA analysis on the exosomes of this invention and unexpectedly demonstrated that these exosomes possessed essentially the same miRNA profile as exosomes derived from natural human breast milk.

[0253] In particular, it was unexpectedly discovered that the exosomes of the present invention contain miRNAs known in the literature to be present in exosomes derived from natural human breast milk and to be essential for infant health and development. These miRNAs include miR-148a-3p, miR-22-3p, miR-125b-5b, and miR-6126.

[0254] Therefore, in some embodiments, the present invention provides a human breast milk exosome, wherein the exosome comprises one or more miRNAs selected from miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

[0255] In some implementations, the exosome comprises miR-148a-3p, miR-22-3p, miR-125b-5b, and miR-6126.

[0256] In some embodiments, the exosome contains one or more miRNAs described in any part of this document at a concentration substantially the same as that of the same one or more miRNAs in exosomes derived from natural human breast milk.

[0257] In some embodiments, the exosome contains one or more miRNAs described in any part of this document at a concentration different from the concentration of the same one or more miRNAs in exosomes derived from natural human breast milk. In some embodiments, the one or more miRNAs can be present at a higher concentration than the concentration of the one or more miRNAs in exosomes derived from natural human breast milk. In some embodiments, the one or more miRNAs can be present at a lower concentration than the concentration of the one or more miRNAs in exosomes derived from natural human breast milk.

[0258] Exosomal lipidomics

[0259] The inventors of this invention performed lipid analysis on the exosomes of this invention and unexpectedly demonstrated that these exosomes possessed essentially the same lipid profile as exosomes derived from natural human breast milk.

[0260] In particular, it was unexpectedly discovered that the exosomes of the present invention contain lipid classes known in the literature to be present in exosomes derived from natural human breast milk and essential for infant health and development. These lipids include cholesterol esters, ceramides, triglycerides, diglycerides, lysophospholipids, total phospholipids, alkyl phospholipids, and alkenyl phospholipids.

[0261] Among these key lipid classes, phospholipids are particularly considered essential for the structure and function of exosomes. It was also unexpectedly discovered that the exosomes of this invention contain phospholipid subclasses known in the literature to be present in exosomes derived from natural human breast milk. These phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0262] Therefore, in some embodiments, the present invention provides a human breast milk exosome, wherein the exosome comprises one or more lipids selected from cholesterol esters, ceramides, triglycerides, diglycerides, total phospholipids, alkyl phospholipids and alkenyl phospholipids.

[0263] In some embodiments, the exosome contains cholesterol esters, ceramides, triglycerides, diglycerides, total phospholipids, alkyl phospholipids, and alkenyl phospholipids.

[0264] In some embodiments, the exosome comprises one or more phospholipids selected from sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0265] In some embodiments, the exosome contains sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0266] In some embodiments, the exosome contains one or more lipids described in any part of this document at a concentration substantially the same as that of the same one or more lipids in exosomes derived from natural human breast milk.

[0267] In some embodiments, the exosome contains one or more lipids described in any part of this document at a concentration different from the concentration of the same one or more lipids in exosomes derived from natural human breast milk. In some embodiments, the one or more lipids can be present at a higher concentration than the concentration of the one or more lipids in exosomes derived from natural human breast milk. In some embodiments, the one or more lipids can be present at a lower concentration than the concentration of the one or more lipids in exosomes derived from natural human breast milk.

[0268] Exosome size

[0269] The inventors of this invention conducted a size analysis on the exosomes of this invention and unexpectedly proved that the diameter of these exosomes is substantially the same as that of exosomes derived from natural human breast milk.

[0270] Therefore, in some embodiments, the present invention provides a human breast milk exosome, wherein the diameter of the exosome is 50 nm to 100 nm, preferably 60 nm to 80 nm, and more preferably 65 nm to 75 nm.

[0271] In some implementations, the diameter of the exosome is 65 nm to 75 nm.

[0272] It should be understood that any feature of exosomes described in any part of this document can be combined. For example, features associated with the exosomal proteome can be combined with features associated with miRNA profiles, lipid profiles, and / or size profiles.

[0273] Treatment

[0274] The exosomes or compositions of the present invention (e.g., pharmaceutical compositions) can be used in therapies, such as for treating diseases or disorders. A method of treating a disease or disorder is also provided, comprising administering a therapeutically effective amount of the exosomes or compositions (e.g., pharmaceutical compositions) as described herein to a subject in need.

[0275] It has been shown that the exosomes of the present invention have a protective effect against barrier integrity alterations triggered by pro-inflammatory cytokines on epithelial Caco-2 HTB-37 cells (Example 7). The exosomes of the present invention effectively promote epithelial barrier function and prevent epithelial inflammation. Therefore, the present invention provides a therapy involving administering the exosomes of the present invention or compositions of the present invention (e.g., pharmaceutical compositions) to a subject for the prevention, treatment, or improvement of symptoms associated with inflammation, particularly epithelial inflammation. In some embodiments, the inflammation is intestinal inflammation. In some embodiments, the inflammation is bronchial inflammation. In some embodiments, the inflammation is respiratory tract inflammation.

[0276] Therefore, this document provides exosomes or compositions of the present invention (e.g., pharmaceutical compositions) for the prevention, treatment, or improvement of symptoms associated with inflammation, particularly epithelial inflammation. A method for the prevention, treatment, or improvement of symptoms associated with inflammation (particularly epithelial inflammation) is also provided, comprising administering a therapeutically effective amount of the exosomes or compositions (e.g., pharmaceutical compositions) described herein to a subject in need. In some embodiments, the inflammation is intestinal inflammation. In some embodiments, the inflammation is bronchial inflammation. In some embodiments, the inflammation is respiratory tract inflammation.

[0277] Inflammation is the response of living tissue to injury or infection. The exosomes of this invention or the compositions of this invention (e.g., pharmaceutical compositions) can also be used to treat inflammation-related diseases (inflammatory disorders).

[0278] Inflammatory diseases include those of the digestive tract, such as esophagitis (inflammation of the esophagus, such as esophageal ulcers), gastroenteritis (inflammation of the stomach and intestinal mucosa, such as gastritis, duodenal ulcers, ileitis, or enterocolitis), colitis (inflammation of the colon, such as diverticulitis), cholangitis (inflammation of the bile ducts), appendicitis (inflammation of the appendix), inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), and irritable bowel syndrome. Inflammatory diseases also include respiratory tract inflammatory diseases and bronchial inflammatory diseases, such as acute respiratory distress syndrome and respiratory infections.

[0279] The exosomes or compositions of the present invention (e.g., pharmaceutical compositions) can be used to increase cell proliferation of epithelial cells (e.g., intestinal cells), reduce their apoptosis and cell death, and alleviate inflammation. A method for increasing cell proliferation of epithelial cells (e.g., intestinal cells), reducing their apoptosis, and alleviating inflammation is also provided, comprising administering a therapeutically effective amount of the exosomes or compositions (e.g., pharmaceutical compositions) as described herein to a subject in need.

[0280] The exosomes or compositions of the present invention (e.g., pharmaceutical compositions) can be used to restore intestinal barrier function in a subject. Therefore, a method for restoring intestinal barrier function in a subject is also provided, comprising administering a therapeutically effective amount of the exosomes or compositions (e.g., pharmaceutical compositions) as described herein to a subject in need.

[0281] As used herein, the term "subject" refers to an animal, particularly a mammal, or a human. Preferably, the subject is a human. In some embodiments, the subject is selected from premature infants, infants, toddlers, children, and adults. Preferably, the subject is selected from premature infants, infants, toddlers, and children.

[0282] Additional embodiments of the present invention

[0283] Provided:

[0284] S1. An in vitro method for producing mammalian breast milk exosomes, the method comprising:

[0285] A) Culture mammary epithelial cells in a culture medium to generate lactating mammary gland-like organoids; and

[0286] B) The lactating cells secrete mammalian milk-like products, and

[0287] C) Purify the exosomes from the mammalian milk product to remove impurities, optionally by chromatography, filtration or ultracentrifugation, to separate the exosomes.

[0288] S2. The method according to statement 2, wherein the duration of step A) is no more than 14 days, optionally 14 days.

[0289] S3. The method according to statement 1 or 2, wherein the lactating cell mammary gland-like organoids from step A) express one or more mammary gland-positive cell markers, optionally selected from KRT-18 and ER.

[0290] S4. The method according to any one of statements 1 to 3, wherein step A) further comprises:

[0291] i) Culture the mammary epithelial cells, and

[0292] ii) Inducing milk protein expression.

[0293] S5. The method according to any one of statements 1 to 4, wherein the lactating cell mammary gland-like organoids from step A) have increased mRNA expression of one or more milk bioactivity markers after induction compared to before induction.

[0294] S6. The method according to any one of statements 1 to 5, wherein the lactating cell mammary gland organoids from step A) have increased mRNA expression of LTF after induction compared to before induction.

[0295] S7. The method according to any one of statements 1 to 6, wherein the lactating cell mammary gland organoids from step A) have increased mRNA expression of MFGE8 after induction compared to before induction.

[0296] S8. The method according to any one of statements 1 to 7, wherein the culture medium is in a suitable 3D culture system, such as MammoCult medium under 3D suspension conditions.

[0297] S9. The method according to statement 8, wherein step Ai) lasts for no more than 7 days, and optionally for 7 days.

[0298] S10. The method according to statement 8 or 9, wherein step Aii) lasts for no more than 7 days, and optionally for 7 days.

[0299] S11. The method according to any one of statements 1 to 10, wherein step A) further comprises:

[0300] i) The mammary epithelial cells were cultured for 7 days in complete MammoCult medium containing the basal medium, proliferation supplement, and supplemented with heparin and hydrocortisone.

[0301] ii) Milk protein expression was induced by incubating the cells in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days.

[0302] S12. The method according to any one of statements 1 to 10, wherein step A) further comprises:

[0303] i) The mammary epithelial cells were cultured for 7 days in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, and heparin.

[0304] ii) Milk protein expression was induced by incubation for 7 days in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0305] S13. The method according to any one of statements 1 to 12, wherein step C) further comprises processing the modified milk-like product to generate a modified mammalian milk-like product.

[0306] S14. The method according to any one of statements 1 to 13, wherein the mammary epithelial cells are human mammary epithelial cells.

[0307] S15. The method according to any of the foregoing statements, wherein step C) further comprises formulating the isolated exosomes into a powder form, optionally by spray drying or freeze drying, or into a liquid form.

[0308] S16. The method according to any of the foregoing statements, wherein step C) further includes sterilizing the isolated exosomes.

[0309] S17. The method according to any of the foregoing statements, wherein step C) further includes storing the isolated exosomes at a temperature below freezing, optionally at -80°C.

[0310] S18. The method according to any of the foregoing statements, the method further comprising formulating the isolated exosomes together with supplemental nutritional ingredients.

[0311] S19. The method according to any of the foregoing statements, wherein the isolated exosomes are dispensed into containers for consumption.

[0312] S20. Mammary gland breast milk exosome product, said mammary gland breast milk exosome product being obtainable according to any of the methods described above, optionally human breast milk exosomes.

[0313] S21. A method for producing mammalian milk fortifier, the method comprising performing the method according to any one of statements 1 to 19.

[0314] It should be understood that the various aspects and embodiments of the invention disclosed herein are examples of specific ways of making and using the invention, and these contents do not limit the scope of the invention when considered in conjunction with the claims and the detailed description herein. It should also be understood that features of various aspects and embodiments of the invention may be combined with other features of the same or different aspects and embodiments of the invention.

[0315] As used in the detailed description and the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the context clearly specifies otherwise. Attached Figure Description

[0316] Figure 1 The diagram illustrates the differentiation of human induced pluripotent stem cells (hiPSCs) using an alternative to the scheme outlined in Ying Qu's paper and as described in step A) of the method of the present invention.

[0317] Figure 2 The diagram illustrates the differentiation of human induced pluripotent stem cells (hiPSCs) according to step A).

[0318] Figure 3 : Shows according to Figure 2 The method used to produce three-dimensional organoid cultures of hiPSCs highly permitted mammary gland differentiation. The mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8, and CSN2 for the 3D differentiation (42-day) protocol is shown. Markers from left to right: pluripotency stage (Nanog), cell line (ectoderm and endoderm) (TUBB3, FOXA2), basal cell / myoepithelial markers (TP63, KR-14), luminal epithelial markers (EpCAM, KRT8), and milk protein (CSN2 (casein β)).

[0319] Figure 4 : This shows a two-dimensional organoid culture of hiPSCs produced as a comparative example. The mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8, and CSN2 for the 2D differentiation (31-day) protocol is shown. Markers from left to right: pluripotency stage (Nanog), cell line (ectoderm and endoderm) (TUBB3, FOXA2), basal cell / myoepithelial markers (TP63, KR-14), luminal epithelial markers (EpCAM, KRT8), and milk protein (CSN2 (casein β)).

[0320] Figure 5 The following diagram illustrates lactation induction in three-dimensional (3D) cultures of mammary epithelial cells. a) Schematic diagram of the culture protocol and lactation induction of mammary epithelial cells (days 7–14). b) Analysis of mammary gland markers keratin 18 and estrogen receptor by flow cytometry during the proliferation phase of mammary epithelial cell cultures. c) RNA expression (ΔCt) levels of human lactoferrin (LTF) and milk fat globule-EGF factor 8 (MFGE8) / lactohemagglutinin during the proliferation (day 7) and lactation induction (day 14) phases. In the panel demonstration, the ΔCt method was used to rank LTF and MFGE8 genes by calculating the mean standard deviation (SD) based on the relative expression of candidate reference genes. Designated genes with the lowest SD were identified as the most stable or most expressed genes.

[0321] Figure 6 The image shows the expression (ah) of different mammary epithelial markers in mammary epithelial cells using gene expression profiling analysis with NanoString technology.

[0322] Figure 7 The median particle size (nm) of EVs purified from epithelial cells and iPSC cells is shown compared to human milk.

[0323] Figure 8 The diagram illustrates different mammary gland differentiation protocols. The protocols outline different procedures for producing mammary gland progenitor cells: within 42 days (a), using bone morphogenetic protein 4 (BMP4) (b), retinoic acid (RA) (c), a combination of BMP4 and RA (d), and a combination of them within a shortened timeframe (31 days) (e). Red boxes indicate the time periods when BMP4 and / or RA are added.

[0324] Figure 9 The combined effects of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) on mammary gland differentiation of induced pluripotent stem cells (iPSCs) using a 3D organoid model are illustrated. *Flow cytometry quantification of the number of mammary gland-positive progenitor cells using several identified markers: EpCAM, CD49f, MUC1, and GATA3, during the differentiation time series (day 25 (for the control protocol) and day 20 (for the shortened time series)) and the pre-induction phase (day 35 (for the control protocol including BMP4 and RA) and day 26 (for the shortened time series)). *A shortened modified protocol (day 31) including the combination of BMP4 and RA is described.

[0325] Figure 10 : This shows the expression (ah) of different mammary epithelial markers derived from iPSCs in lactating cells in both the normal length and shortened protocols.

[0326] Figure 11 This study demonstrates a targeted proteomic analysis of osteopontin protein secretion based on liquid chromatography-mass spectrometry (LC-MS / MS). Osteopontin secretion was detected in iPS-derived mammary gland cell cultures using either a control differentiation method or a combination of bone morphogenetic protein 4 (BMP4), retinoic acid (RA), and the combination of these two factors.

[0327] Figure 12: a, Venn diagram showing the number of proteins identified only in EVs purified from conditioned medium (days 39 to 41) (left circle), the number of proteins identified only in EVs purified from human milk (right circle), and the number of proteins identified in both types of EVs (overlapping circles); b, table showing a list of the top 50 proteins (based on FDR) detected in both types of EVs (human milk and conditioned medium).

[0328] Figure 13: a. Venn diagram showing the number of miRNAs significantly downregulated in EVs purified from conditioned medium (days 39–41) compared to human milk EVs (circles); the number of miRNAs significantly upregulated in conditioned medium EVs compared to human milk EVs (squares); and the number of miRNAs not expressed differentially in the two types of EVs (overlapping circles and squares). b. Venn diagram showing the results obtained by subsequently repeating the experiments of Example 6 and using two additional batches of cell-based EVs. Venn diagram showing the number of miRNAs significantly downregulated in EVs purified from conditioned medium (days 39–41) compared to human milk EVs (circles); the number of miRNAs significantly upregulated in conditioned medium EVs compared to human milk EVs (squares); and the number of miRNAs not expressed differentially in the two types of EVs (overlapping circles and squares). c. Table showing a list of the top 4 miRNAs (based on FDR) expressed in both types of EVs (human milk and conditioned medium).

[0329] Figure 14 The chart shows the proportions of lipid classes (left) and phospholipid classes (right) found in EVs purified from human milk and conditioned medium (days 39–41). Data are expressed as percentages of total lipids and total phospholipids, respectively. TG: triglycerides; DG: diglycerides; PL: total phospholipids; Lyso: lysophospholipids; alkyl: phospholipid-O; alkenyl: phospholipid-P; CE: cholesterol esters; CER: ceramides; SM: sphingomyelin; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine. Specific lipids and phospholipids in the bar charts, from bottom to top, correspond to the lipids or phospholipids in the abbreviation list, from left to right.

[0330] Figure 15 The median particle size (nm) of EVs purified from iPSCs (conditioned medium (day 41)) and human milk, as analyzed by nanoflow cytometry, is shown.

[0331] Figure 16 The effects of treating Caco-2 monolayers with dexamethasone (1 µM), human milk-derived EVs (HM EVs) (1.107 EVs / well), and lactocyte-derived EVs (1.107 EVs / well) before application of IFNγ and TNFα are shown. The control is Caco-2 cells without any attack.

[0332] Figure 17The results show the effects of treating Caco-2 monolayers with dexamethasone (1 µM), human milk-derived EVs (HM EVs) (1.107 EVs / well), and lactocyte-derived EVs (1.107 EVs / well) before application of IFNγ and TNFα. Numerical values ​​indicate the effect after 48 hours of inflammatory challenge. The control group consisted of Caco-2 cells without any challenge.

[0333] Experimental Section

[0334] Example 1

[0335] hiPSCs were cultured and differentiated into lactating cells to obtain human milk-like products.

[0336] According to the process described by Ying Qu et al. in Stem Cell Report (Vol. 8, pp. 205-215, February 14, 2017), lactating cells are cultured starting from ihPSCs to collect the human milk-like product secreted therefrom, which can be used for treatment and / or as a breastfeeding alternative according to the present invention.

[0337] Example 2

[0338] hiPSCs were cultured and differentiated into 3D lactogenic cells to obtain human milk-like products.

[0339] In steps A) and B) of the method according to the invention, lactating cells are cultured starting from hiPSCs, thereby collecting the human milk-like product secreted therefrom, which can be used for treatment and / or as a breastfeeding alternative according to the invention.

[0340] Example 3

[0341] Another alternative method is to culture and differentiate hiPSCs into lactating cells to obtain human milk-like products.

[0342] Lactobacillus differentiation can be efficiently obtained from hiPSCs under alternative culture conditions, including conditions 1 through 4 as described below:

[0343] 1. 2D cultures of monolayer cells on culture dishes coated with spheronin were derived from EB and cultured for at least 28 days in a medium containing the following components: RPMI 1640 containing L-glutamine; fetal bovine serum (FBS); insulin; epidermal growth factor (EGF); hydrocortisone; and a penicillin / streptomycin mixture (penicillin / streptomycin: antibiotic-antifungal solution).

[0344] 2. 2D cultures of adherent cell aggregates (EB) on culture dishes coated with spheronin were derived from EB and cultured for at least 28 days in a medium containing the following components: RPMI 1640 containing L-glutamine; fetal bovine serum (FBS); insulin; epidermal growth factor (EGF); hydrocortisone; penicillin-streptomycin mixture (antibiotic-antifungal solution).

[0345] 3.3D cultures were suspended in MammoCult medium for at least 10 days, then cultured for another 5 days in a specific medium (e.g., EpiCultB) in the presence of parathyroid hormone, in a mixed floating gel (e.g., Matrigel and type 1 collagen), and then cultured for another 25 days in the presence of insulin, HGF, hydrocortisone and FGF10.

[0346] 4. 3D cultures of EB were cultured in suspension in MammoCult medium (ultra-low adhesion plates) for at least 10 days, then in suspension medium in the presence of parathyroid hormone for another 5 days, and then in insulin, HGF, hydrocortisone and FGF10 for another 25 days.

[0347] Example 4

[0348] 2D and 3D lactocyte differentiation based on human induced pluripotent stem cell (hiPSC) line 603

[0349] (a) 3D lactocyte differentiation based on human induced pluripotent stem cell (hiPSC) line 603 :

[0350] Human induced pluripotent stem cell (hiPSC) line 603 was used for the differentiation of 3D lactocytes. Human induced pluripotent stem cell (hiPSC) line 603 was purchased from Fujifilm Cellular Dynamics (FCDI).

[0351] (i) For the 3D differentiation protocol according to the invention, hiPSC single cells were incubated overnight in E8 medium containing 10 μM ROCK inhibitor to form EB (spheres).

[0352] The next day, the culture medium was replaced with E8 (from day -2 to day 0).

[0353] The following day, the medium was replaced with Mammo1 medium (MammoCult medium with proliferation supplement, heparin (4 µg / mL) and hydrocortisone containing penicillin / streptomycin (0.48 µg / mL)) for 10 days (day 0 to day 10). The medium was changed every two days.

[0354] (ii) After culturing in Mammo2 medium (EpiCultB+ supplement, 100 ng / ml pTHrP, plus penicillin / streptomycin) for 5 days, perform the above differentiation. (Change the medium every 3 days from day 10 to day 15)

[0355] (iii) To induce branched epithelial structure, vesicle differentiation, and mammary cell specialization, mEBs (globules / mammary globules) were fed with Mammo3 medium (complete EpiCultB, hydrocortisone (1 µg / ml), insulin (10 µg / ml), FGF10 (50 ng / ml), HGF (50 ng / ml), and penicillin / streptomycin) for 20 days. The medium was changed every 3 days (from day 15 to day 35).

[0356] (iv) Finally, to induce the production of bioactive substances in milk (3D), cells were cultured for 7 days in Mammo4 medium (complete EpiCultB, 10% FBS, prolactin (10 µg / ml), hydrocortisone (1 µg / ml), insulin (10 µg / ml), progesterone, β-estradiol, and penicillin / streptomycin), with the medium changed every 3 days (from day 35 to day 42). Throughout differentiation, the spheres were maintained in suspension culture (with shaking at 95 rpm). Differentiation ended on day 42. Results were presented in… Figure 3 As shown in the image.

[0357] (b) 2D lactocyte differentiation based on human induced pluripotent stem cell (hiPSC) line 603

[0358] Human induced pluripotent stem cell (hiPSC) line 603 can also be used for the differentiation of 2D lactocytes. Human induced pluripotent stem cell (hiPSC) line 603 was purchased from Fujifilm Cellular Dynamics (FCDI).

[0359] For the 2D differentiation protocol (used for comparison), lactose-containing medium (RPMI 1640, 20% FBS, 1 mM glutamine, 4 µg / ml insulin, 20 ng / ml EGF, 0.5 µg / ml hydrocortisone containing penicillin / streptomycin) was used at all differentiation stages. Cells were incubated at 37°C and 5% CO2. The medium was changed every two days. Results were presented in... Figure 4 As shown in the image.

[0360] (c) Results

[0361] Using quantitative RT-PCR ( Figure 3 3D differentiation Figure 4The study captured different differentiation stages during lactation cell derivation using a 2D differentiation model. NaNog expression, a marker of pluripotency, was reduced in both 2D and 3D settings, coinciding with cell maturation and differentiation. Neuroectodermal and endoderm markers TUBB3 (tubulin β3 class III) and forkhead box protein A2 (FOXA2) were not significantly expressed in the 3D format, and an increase in TUBB3 was only captured in the 2D setting. This suggests that hiPSCs pattern towards non-neuroectodermal cell lines, thus enriching mammary progenitor cells in the 3D format. The inventors investigated the expression patterns of commonly used basal cell / myoepithelial markers such as p63 (p53-homogeneous nucleoprotein) and cytokeratin 14 (KRT-14). Both markers were clearly detectable in both systems. Additionally, epithelial cell adhesion molecule (EpCAM) and cytokeratin 8 (KRT8) were tracked only in the 3D system, and KRT8 was only partially expressed in the 2D format. Therefore, the 3D platform in the organoid setting expressed common markers of mammary tissue, lumen, and basal structure. These mammary-like organoids expressed human breast milk-specific proteins, including CSN2 (casein β), lactin peptides, and hormone receptors. Luminal cells specifically expressed EpCAM, MUC1, CD49F, GATA3, CK8, and CK18, while basal cells specifically expressed CK14, α-smooth muscle actin, and P63. Ultimately, EpCAM and CD49F double-positive cells were detectable at an earlier progenitor stage between day 10 and day 35. Interestingly, CSN2 expression was captured only at the final time point (day 42) in the 3D organoid system and not in the 2D guided differentiation platform.

[0362] As described below, analysis of mammary gland-like organoid secretor bodies revealed the secretion of human milk-specific bioactive substances, including oligosaccharides (including lactose and some HMOs), lipids (including four fatty acids), proteins (seven detected, including casein), and miRNAs (75 detected, including 11 commonly detected in HBM).

[0363] The presence of lactose or human milk oligosaccharides (HMOs) in primary cell supernatants was analyzed with minimal modifications according to the procedure described in “Austin and Benet, Quantitative determination of non-lactose milkoligosaccharides, Analytica Chimica Acta 2018, 1010, 86-96”. Samples were analyzed using UHPLC, and lactose or HMOs (HMOs) were quantified using a calibration curve for lactose and a mixture of seven HMOs (2'FL, 3FL, DFL, LNT, LNnT, 3'SL, and 6'SL). The estimated limit for the above method was 0.1 mg / L. In primary cell supernatants, lactose (0.22 mg / L) and 6'SL (0.32 mg / L) were detected on day 42.

[0364] Fatty acids were analyzed in culture medium and cell supernatants by gas chromatography coupled with a flame ionization detector. Specifically, the supernatant obtained on day 42 was analyzed to investigate the presence of fatty acids in several lipids. A 7890A gas chromatograph equipped with a 7693 autosampler and a fused silica CP-Sil 88 capillary column (100% cyanopropyl polysiloxane) was used. This autosampler was equipped with a preparative site module; a 100 μm, 0.25 mm ID, 0.25 mm membrane thickness was used, along with a split injector heated to 250 °C (1:25 ratio) and a flame ionization detector operated at 300 °C. Fatty acid methyl esters (FAMEs) were prepared by direct transesterification of the samples with methanolic chloric acid. FAMEs were separated using capillary gas chromatography (GC) with FID. FAME identification was performed by retention time (RT) and compared with an external standard. Fatty acid quantification was performed using methyl C11:0 as an internal standard. TAG C13:0 was used as a second internal standard to control the transesterification performance of the above method. After adding the internal standard, the solution was mixed with 2 mL of methanol, 2 mL of methanol / HCl (3N), and 1 mL of hexane. The mixture was heated at 100 °C for 60 minutes, then allowed to cool to room temperature (approximately 15 minutes), and the reaction was stopped by adding 2 mL of water. After centrifugation, the organic phase was directly injected into the GC.

[0365] The fatty acid results from the protocol of Example 4a on day 42 are reported in Table 1 (observed differences between the culture medium and the supernatant).

[0366] The fatty acids expressed in the cell supernatant samples are listed in Table 1 below.

[0367]

[0368] Proteins in the cell supernatant were analyzed using SDS-PAGE characterization, followed by LC-MSMS analysis of the gel strips used for identity verification. For SDS-PAGE analysis, the total volume of the prepared sample was loaded onto the gel. Human milk samples were added as a control for comparison. Selected gel regions (strips) were cut using LC-MSMS to examine human proteins. Finally, the strips were submitted for in-gel trypsin digestion and analyzed by LC-MSMS. LC-MSMS data were analyzed using Peaks Studio and matched against UniProt's human protein database.

[0369] Table 2 below lists the best candidates for all excised adhesive strips.

[0370]

[0371] Exosome isolation and miRNA characterization were performed using an ExoQuick polymer network. ExoQuick polymers precipitate exosomes by forming a network and collecting all exosomes of a given size. Once the ExoQuick network is formed, the exosomes can be easily precipitated into globules using a simple, low-speed centrifugation method. The exosomes are intact, ready for protein or RNA analysis, and biologically active for functional studies. Precipitation buffer was added to the sample at a ratio of 0.25x, followed by vortexing. The mixture was incubated overnight at 4°C. After incubation, the sample was centrifuged at 1,500 x g for 30 minutes. The exosome globules were resuspended by vortexing in an initial volume of either QC buffer XE (QIAGEN) or miRNA Whole Transcriptome Assay lysis buffer from HTG EdgeSeq for miRNA characterization. To assess extracellular vesicle (EV) isolation, the supernatant was first centrifuged at 3000 g for 15 minutes to remove globules and debris. Then, precipitation was performed overnight at 4°C using 100 µL of culture medium and ExoQuick buffer (0.25-fold ratio). The EV precipitate was recovered by centrifugation at 1500 g for 30 min. Each sample was precipitated twice: the first EV precipitate was resuspended in buffer XE (QIAGEN) for further possible analysis, and the second precipitation was performed only in 50 µL of HTG lysis buffer to reduce the concentration by 10-fold before miRNA characterization analysis via HTG.

[0372] For miRNA characterization, the sample was used directly in the first step of lysis. Therefore, whole samples were used directly and lysed with plasma lysis buffer at a 1:1 ratio. Next, proteinase K (1 / 10) was added, and the sample was incubated at 50°C and 600 rpm for 3 hours on a Thermomixer. EVs were resuspended in lysis buffer and lysed under the same conditions, with an additional step of incubation at 95°C for 10 minutes before the lysis incubation. Following the V2 process of the miRNA Whole Transcriptome Assay from HTG EdgeSeq, 26 µl of the lysate was processed on an HTG processor using 70 µl of oil. For indexing and amplification libraries, the sample was labeled with Illumina adaptors and processed using OneTaq. ® HotStart 2X Master Mix GC buffer (95°C–4 min; 16 cycles: 95°C–15 s, 56°C–45 s, 68°C–45 s; 68°C–10 min; hold at 4°C) was indexed by PCR and cleaned with AMPure (2.5 ratio) on a robotic liquid processor SciClone NGS workstation (Perkin Elmer). Pools were obtained using a custom pooling procedure on a Hamilton robot. Samples were pooled using GX Touch Chip HS quantification. Pools were then manually purified a second time using AMPure magnetic beads (1.8 ratio) to remove any trace primer dimers that might remain, and the pools were quantified with Qubit to adjust the final concentration to 2 nM. As a final step, for MiSeq sequencing, pools were loaded onto MiSeq at 20 pM and a 5% PhiX peak, and sequencing was performed against 50-base single reads on MiSeq using a 150V3 kit.

[0373] In short, 974 miRNAs were detected in the cell supernatant, of which more than 75 were miRNAs highly expressed in the milk sample.

[0374] Table 3 below lists the top ten highly expressed miRNAs.

[0375]

[0376] The findings of this study provide a novel 3D organoid model based on iPSCs for studying the fate and function of normal mammary cells and how the production of bioactive substances in breast milk is regulated and developed.

[0377] Example 5

[0378] Using differentiation medium M1 / M4, mammary epithelial cells were cultured in a 3D format with and without matrix for 7 days of proliferation and 7 days of induction phase. Figure 5 a). Other differentiation media can also be used. Simply put, 1-5 million dissociated epithelial cells are seeded onto a flat shaker platform in a 6-well plate (ultra-low adsorption) containing 4.5 mL of medium. Quantitative analysis using flow cytometry shows that the cultured cells express mammary gland-specific markers such as keratin 18 and estrogen receptor (…). Figure 5 b), and is able to express mRNA levels of human lactoferrin (LTF) and milk fat globule-EGF factor 8 (MFGE8) or lactoglossin (b) during the proliferation phase (day 7) and induction phase (day 14). Figure 5 c, Figure 5 d).

[0379] Using NanoString technology for gene expression profiling, we assessed the expression of different breast epithelial markers in breast epithelial cells. Figure 6 The expression of the mammary epithelial progenitor cell marker CD24 was consistent and showed a trend toward higher expression during lactation. Figure 6 a). Interestingly, it was observed that as lactation progressed, cells showed a decrease in the expression of mammary basal-like cell markers such as KRT5, KRT14, and ITGA6 (CD49f) (a). Figure 6 bd). It appears that cells maintain a lumen-like phenotype during and after lactation using different markers (such as EpCAM, KRT8, KRT18, and MUC1). Figure 6 The late induction phase is characterized by lactogenic cell (lumen-like cell)-specific secretory profiles that elicit different proteins, such as lactoferrin (LTF), clustering proteins, and fatty acid synthase (FASN) (Table 1a). Exosomes isolated from purified epithelial cells post-lactation also showed high expression of milk-specific miRNAs, listed in Table 1b. Expression of mammary epithelial markers in post-lactation mammary epithelial cells and milk-specific miRNAs in exosome-purified epithelial cells were also observed using alternative differentiation conditions.

[0380] Table 1 :

[0381] a

[0382]

[0383]

[0384] b

[0385]

[0386] Flow cytometry analysis of EVs isolated from epithelial cells was performed using a NanoFCM instrument, and the results were compared with those of exosomes derived from natural breast milk as a control. Examples of the results are shown in... Figure 7 The figure shows that the size range of epithelial EVs is similar compared to the control (see [reference]). Figure 7 ).

[0387] Further protein expression analysis and NanoFCM antibody labeling were performed on the isolated EVs to quantify the expression of the tetraspan membrane proteins CD9, CD63, and CD81. The mean expression level of these proteins was 45%, indicating a significant expression profile with a high confidence level.

[0388] Example 6

[0389] To generate milk bioactive substances highly similar to those found in human breast milk, different methods using bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) were established on a 3D platform using iPSC-derived mammary gland organoids as biomimetic models of human mammary glands at 42 and 31 days (as a shortened time-course protocol). Figure 8 ).

[0390] The expression of mammary gland progenitor cell markers (such as EpCAM (CD326), CD49f, MUC1 (CD227), and GATA3) in the 42-day regimen and the shortened time-series regimen (31 days – *describes 3D-iPSC cells in the shortened modified regimen (31 days) including the combination of BMP4 and RA) in single or combined forms, compared with control conditions, can be quantified by flow cytometry. Figure 9 ac) and post-induction phase ( Figure 9 During the df period, their expression profiles are maintained.

[0391] We evaluated the expression of different mammary epithelial markers in lactating cells derived from iPSCs in both normal length and shortened protocols. Figure 10 This shortened protocol induces RELA as a specific gene used to identify mammary epithelial cell lineages during iPSC differentiation. Figure 10 a). In the shortened scheme ( Figure 10 In the be) regimen, in addition to mature luminal markers such as GATA3, specific lactogenic cell markers such as EpCAM and KRT8 / 18 were induced. Furthermore, when we switched to the 31-day regimen, the induced ESRRA level ( Figure 10f). Similarly, shortened differentiation process is associated with elevated levels of progenitor cell markers in mammary glands, such as CD24 and ITGA6 (CD49f). Figure 10 gh).

[0392] Human osteopontin peptides -GDSVVYGLR and -YPDAVATWLNPDPSQK were specifically detected in cell culture supernatant by LC-MS / MS. Figure 11 ).

[0393] Example 7

[0394] A more systematic analysis of a specific human breast milk bioactive component (i.e., exosomes) produced through step A revealed that its compositional profile was very close to that of exosomes from a positive control of human breast milk.

[0395] Human iPSCs were cultured and aggregated in 3D using ultra-low adsorption 6-well plates. Embryoids were formed by continuous rotation and shaking, followed by a multi-step differentiation protocol (41 days) to generate functional mammary gland organoids. During the last 7 days of the differentiation protocol (days 35 to 41), the mammary gland organoids were placed in a specific medium supplemented with prolactin and growth factors to induce the secretion of milk-specific bioactive compounds. This medium was changed every other day (days 35, 37, and 39).

[0396] Conditioned media were collected and combined on days 39 and 41 of step A (this corresponds to the media in contact with cells from day 37 to day 41). EVs were purified from the conditioned media and skimmed human milk (available from Lee Biosolutions, 991-01-P, collected from milk of at least 5 lactating women after week 4 of lactation) by ultracentrifugation.

[0397] Analysis of EV-related protein profiles by liquid chromatography-mass spectrometry (LC-MS) showed approximately 30% to 50% overlap when comparing breast milk control EVs and cell-derived EVs. Figure 12a ). Figure 12bThe results show that most proteins highly expressed in breast milk exosomes are also expressed in cell-based exosomes, including lactoferrin (LTF), annexin A2 (ANXA2), and lactoglucin (MFGE8), as well as Muc1, CD9, CD81, and CD63. Interestingly, some proteins such as C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2) were not detected in cell-based EVs but were detected only in breast milk EV positive controls.

[0398] The experiment was then repeated using two additional batches of cell-based EVs, further confirming that the proteins hypoxia upregulation protein 1 (HYOU1), apolipoprotein A-IV (APOA4), and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) were not detected in cell-based EVs, but were only detected in the positive control of breast milk EVs. Furthermore, in one of the two additional batches of cell-based EVs, the proteins C4a anaphylatoxin (CFA), cytoplasmic threonine-tRNA ligase 1 (TARS1), and cytoplasmic nonspecific dipeptidase (CNDP2) were also not detected.

[0399] Proteomics analysis can be performed using the following equipment and conditions:

[0400]

[0401] Sequencing analysis of the miRNA profile of EVs using HTG molecular whole transcriptomics revealed a high degree of overlap (95% to 99%) between breast milk exosomes and cell-based exosomes. Figure 13a This includes the detection of validated miRNAs such as miR-148a-3p, miR-22-3p, miR-125b-5b, and miR-6126. Figure 13c The results were then repeated using two additional batches of cell-based EVs. Figure 13b As shown in the image.

[0402] Lipometrix analysis (LC-MS / MS non-targeted lipidomics analysis) was used to analyze the lipid composition of EVs purified from both conditioned medium and human milk, revealing a similar overall lipid class profile, including cholesterol esters, ceramides, triglycerides, diglycerides, total phospholipids, alkyl phospholipids, and alkenyl phospholipids. Figure 14 The same phenomenon was observed when analyzing subclass spectra, such as phospholipid spectra.

[0403] Flow cytometry analysis of EVs isolated from epithelial cells, iPSCs (conditioned medium (step A)), or breast milk controls showed similar sizes (see [link]). Figure 15 ).

[0404] Example 8

[0405] Assessing the tight junction function of intestinal epithelial cells by measuring transepithelial electrical resistance (TEER).

[0406] Materials and methods

[0407] Caco-2 HTB-37 cells were cultured on Transwell membranes for 3 weeks to allow differentiation and the formation of a tight barrier. Cells were seeded at a concentration of 6.72 × 10⁴ cells / well in 12-well insert plates and cultured in DMEM medium supplemented with heat-inactivated fetal bovine serum (10%), non-essential amino acids (1%), and penicillin / streptomycin solution (1%). After 3 weeks, differentiated cells were cultured in exosome-free medium supplemented with PBS (medium), 1 µM dexamethasone, and 1.10⁷ human milk-derived (HM EV) exosomes / well or 1.10⁷ lactocyte-derived exosomes / well (Lactocyte EV). Cells were then subjected to inflammatory attack, including pre-stimulation with interferon (IFN)γ for 18 hours to trigger tumor necrosis factor (TNF) α receptor expression, followed by TNFα stimulation for 48 hours. Transepithelial resistance (TEER) was monitored as a marker of epithelial barrier integrity.

[0408] result

[0409] Figure 16 This study demonstrates the effects of treating Caco-2 monolayers with dexamethasone (1 µM), human milk-derived EVs (HM EVs) (1.107 EVs / well), and lactating cell-derived EVs (1.107 EVs / well) before application of IFNγ and TNFα. The control group consisted of untreated Caco-2 cells. Figure 17 The effects of treating Caco-2 monolayers with dexamethasone (1 µM), human milk-derived EVs (HMEVs) (1.107 EVs / well), and lactocyte-derived EVs (1.107 EVs / well) prior to the application of IFNγ and TNFα are shown. Numerical values ​​indicate the effects after 48 hours of inflammatory challenge.

[0410] The control group consisted of Caco-2 cells that had not been attacked in any way.

[0411] Human milk EVs exerted a protective effect against barrier integrity alterations triggered by pro-inflammatory cytokines, achieving a protective effect of 43.1% compared to controls (unattacked cells). Lactocyte-derived EVs showed similar efficacy in preventing TEER decline.

[0412] It should be understood that various changes and modifications to the currently preferred embodiments described herein will be apparent to those skilled in the art. These changes and modifications can be made without departing from the spirit and scope of the invention and without diminishing the accompanying advantages. Therefore, such changes and modifications are intended to be covered by the appended claims.

Claims

1. Isolated human breast milk exosomes in which hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4) and procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1) were not detected by liquid chromatography-mass spectrometry.

2. The exosomes according to claim 1, wherein C4a anaphylatoxin (CFA), hypoxia upregulated protein 1 (HYOU1), apolipoprotein A-IV (APOA4), procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1), cytoplasmic threonine-tRNA ligase 1 (TARS1) and cytoplasmic nonspecific dipeptidase (CNDP2) were not detected in the exosomes by liquid chromatography-mass spectrometry.

3. The exosomes according to claim 1 or claim 2, wherein the exosomes comprise lactoferrin (LTF), annexin A2 (ANXA2), lactoglucosin (MFGE8), MUC1, and tetraspan proteins CD9, CD81, and CD63.

4. The exosomes according to any one of claims 1 to 3, wherein the exosomes comprise one or more miRNAs selected from miR-148a-3p, miR-22-3p, miR-125b-5b, and miR-6126.

5. The exosomes according to any of the preceding claims, wherein the exosomes comprise one or more lipids selected from cholesterol esters, ceramides, triglycerides, diglycerides, lysophospholipids, total phospholipids, alkyl phospholipids, and alkenyl phospholipids.

6. The exosome according to any of the preceding claims, wherein the exosome comprises one or more phospholipids selected from sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine.

7. The exosomes according to any of the preceding claims, wherein the diameter of the exosomes is 50 nm to 100 nm.

8. The exosomes according to claim 7, wherein the diameter of the exosomes is 65 nm to 75 nm.

9. The exosomes according to any of the preceding claims, wherein the exosomes have the same function as exosomes derived from natural human breast milk.

10. The exosome according to any of the preceding claims, wherein the exosome has the same structure as exosomes derived from natural human breast milk.

11. An exosome population, wherein the population comprises exosomes according to any of the preceding claims.

12. A composition comprising exosomes or exosome populations according to any of the preceding claims.

13. An in vitro method for producing exosomes according to any one of claims 1 to 10 or populations or exosomes according to claim 11, the method comprising: A) Culture mammary epithelial cells in a culture medium to generate lactating cells, mammary gland-like organoids; as well as B) The lactating cells secrete mammalian milk-like products, and C) Purify the exosomes from the mammalian milk product to remove impurities, optionally by chromatography, filtration or ultracentrifugation, to separate the exosomes.

14. The method of claim 13, wherein the duration of step A) is no more than 14 days, optionally 14 days.

15. The method according to claim 13 or 14, wherein step A) is performed under 3D suspension culture conditions.

16. The method according to claims 13 to 15, wherein step C) further comprises formulating the isolated exosomes into a powder form, optionally by spray drying or freeze drying, or into a liquid form.

17. The method according to claims 13 to 16, wherein step C) further comprises sterilizing the isolated exosomes.

18. The method according to claims 13 to 17, wherein step C) further comprises storing the isolated exosomes at a temperature below freezing, optionally at -80°C.

19. The method according to claims 13 to 18, further comprising formulating the isolated exosomes together with supplemental nutritional ingredients.

20. The method according to claims 13 to 19, wherein the separated exosomes are dispensed into containers for consumption.

21. A human breast milk exosome product, said human breast milk exosome product being obtainable by the method according to any one of claims 13 to 20.

22. A method for producing a human milk fortifier, the method comprising carrying out the method according to any one of claims 13 to 20.

23. The exosomes according to any one of claims 1 to 10, the exosome population according to claim 11, the composition according to claim 12, or the exosome product according to claim 21, for use in a therapy.

24. A method for treating a disease or disorder, the method comprising administering to a subject in need a therapeutically effective amount of exosomes according to any one of claims 1 to 10, a population of exosomes according to claim 11, a composition according to claim 12, or an exosome product according to claim 21.

25. The exosomes according to any one of claims 1 to 10, the exosome population according to claim 11, the composition according to claim 12, or the exosome product according to claim 21, for the prevention, treatment, or improvement of symptoms associated with inflammation.

26. A method for preventing, treating, or improving symptoms associated with inflammation, the method comprising administering to a subject in need a therapeutically effective amount of exosomes according to any one of claims 1 to 10, a population of exosomes according to claim 11, a composition according to claim 12, or an exosome product according to claim 21.

27. The exosomes according to any one of claims 1 to 10, the exosome population according to claim 11, the composition according to claim 12, or the exosome product according to claim 21, used according to claim 23, or the method according to claim 24, wherein the inflammation is intestinal inflammation.

28. The exosomes according to any one of claims 1 to 10, the exosome population according to claim 11, the composition according to claim 12, or the exosome product according to claim 21, used according to claim 23, or the method according to claim 24, wherein the inflammation is bronchial inflammation.

29. The exosomes according to any one of claims 1 to 10, the exosome population according to claim 11, the composition according to claim 12, or the exosome product according to claim 21, used according to claim 23, or the method according to claim 24, wherein the inflammation is respiratory inflammation.

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