Method for producing a milk-like product

Abstract

A method for producing a mammalian milk-like product, e.g., a human milk-like product, comprising generating lactocytes derived from mammalian mammary epithelial cells, e.g., human mammary epithelial cells, and expressing a mammalian milk-like product, e.g., a human milk-like product, from the lactocytes.

Description

[Technical field]

[0001] The present invention relates to a method for in vitro production of a mammalian milk-like product, such as a human milk-like product, comprising generating lactocytes derived from mammalian mammary epithelial cells, such as human mammary epithelial cells, and / or mammary gland-like organoids comprising such lactocytes through culture and differentiation, and expressing a mammalian milk-like product, such as a human milk-like product, from such lactocytes and / or mammary gland-like organoids.The present invention also relates to a mammalian milk-like product, such as a human milk-like product, obtainable from such a method.

[0002] [Background technology] Mammalian milk, particularly human milk, is a complex fluid that contains many components, each of which can contribute substantially to the health of the infant and possibly the mother. It has become clear that human breast milk is the most suitable source of nutrition, at least up to the age of 6 months. Many components of human milk are not found at all, are barely found, or are less active in the cow's milk that is the basis for the manufacture of infant formulas. Such components include, for example, the protein lactoferrin, proliferation / growth factors, long-chain polyunsaturated fatty acids, or oligosaccharides. Human milk compositions are used as the gold standard for developing current infant formulas, but despite recent major developments in infant formula compositions, it is unrealistic to think that a replica of human milk can be achieved with current manufacturing processes.

[0003] Today, the only source of human milk is human donors (lactating mothers). Milk donation has been reported for non-commercial (human milk biobanks) and commercial use. However, milk donation is limited and is subject to strong regulatory, safety, and sometimes ethical or religious constraints.

[0004] Stem cells have been found in mammalian milk, particularly human milk, and are called human breast milk stem cells (HBsCs). hBSCs have been shown to be highly plastic and differentiate in culture into multiple cell types, and more importantly into the three lineages necessary to form the lobulo-alveolar architecture of the human mammary gland (Hassiotou F. et al. Stem Cells. 2012). However, the use of hBSCs to produce human breast milk is neither practical nor sustainable, as it requires human donors.

[0005] A technology based on cell lines with stem cell functions, called induced pluripotent stem cells (iPSCs), is known. A reliable two-step protocol has been developed to generate human mammary-like organoids from human iPSCs (hiPSCs) (Ying Qu et al, Stem Cell Report vol 8, 205-215, February 14, 2017). th , 2017).

[0006] It is therefore an object of the present invention to provide an alternative and improved method for producing mammary gland cells, which recapitulates the expression of mammalian milk, e.g. human milk, in cultured cells without the use of early stage undifferentiated stem cells. It is also an object of the present invention to prepare customized mammalian milk-like products, e.g. human milk-like products, in cultured cells that can be adapted to the specific needs of the recipient and / or to produce human milk bioactives to complement existing bovine-based solutions for infant nutrition, also without the use of stem cells.

[0007] [Summary of the Invention] The present invention solves the above technical problems. Provided herein is a method for producing a mammalian milk-like product, comprising: A) culturing mammary epithelial cells in a culture medium to generate lactocytic mammary-like gland organoids; B) secreting a mammalian milk-like product from said lactocytes.

[0008] Also provided herein is a human milk-like product obtainable according to the methods described herein.

[0009] Also provided herein is a human milk-like product according to the methods described herein for use in therapy.

[0010] Also provided herein is the use of the human milk-like product according to the methods described herein as a human milk replacer, optionally as a breast milk replacer.

[0011] [Mode for carrying out the invention] definition In the context of the present invention, the term "in vitro" means performed or occurring in a test tube, culture dish, bioreactor, or anywhere outside the body of a living organism.

[0012] In the context of the present invention, the term "mammal" refers to animals belonging to the mammalian species, such as humans, cows, monkeys, camels, sheep, goats, etc.

[0013] In the context of the present invention, the term "lactocytes" or "mammary gland-like cells" refers to secretory epithelial cells that express the CK18 cell marker and are derived from mammalian mammary gland epithelial cells, in particular from human mammary gland epithelial cells. Human mammary gland epithelial cells as used herein are commercially available and can be selected from any suitable cell line. Suitable human mammary gland epithelial cell lines in the context of the present invention can be, for example, non-tumorigenic cell lines such as MCF-10 or tumorigenic cell lines such as MCF-7.

[0014] In the context of the present invention, the term "mammary gland-like organoid" or "mammary-like organoid" refers to a miniaturized and simplified version of the mammary gland, developed in two or three dimensions (2D / 3D) and containing lactocytes as defined above.

[0015] In the context of the present invention, the term "human milk-like product" refers to a cell-cultured milk product. Such a product is an edible product expressed by the lactocytes and / or mammary gland-like organoids produced according to the methods of the present invention.

[0016] The "human milk-like product" according to the present invention can have the same components (e.g., in terms of bioactives, macronutrients and micronutrients and their levels) as human breast milk from a well-nourished mother, which is referred to herein as a "standardized human milk product." Alternatively, the "human milk-like product" according to the present invention can have different component ratios and concentrations than those naturally found in human breast milk from a well-nourished mother, which is referred to herein as a "non-standardized milk-like product." The "human milk-like product" according to the present invention can be modified to include components that are not naturally found in human breast milk from a well-nourished mother ("modified milk-like product"). Non-limiting examples of human milk-like products are selected from the group consisting of supplements, fortifiers, human breast milk substitutes (or replacements), and ingredients enriched in only one and / or some of the bioactives, macronutrients and micronutrients that can typically be found in human breast milk from a well-nourished mother.

[0017] "Human milk-like products" can be used in place of ingesting naturally secreted milk ("human milk substitutes"). Milk substitute products can be used as supplements ("human milk supplements") or fortifiers ("human milk fortifiers") to be ingested in combination with naturally secreted milk.

[0018] In one embodiment, the standard human milk-like product according to the invention comprises at least the macronutrients and micronutrients that can typically be found in human breast milk from well-nourished mothers. In one embodiment, the human milk-like product according to the invention comprises proteins, peptides, 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, myo-inositol and L-carnitine. In one embodiment, the human milk-like product according to the invention also comprises at least one bioactive substance selected from the group consisting of growth / growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and / or exosomes), exosome-derived bioactive substances (e.g. miRNA) and secretory IgA. The standard human milk-like product according to the present invention is not a naturally occurring human breast milk secretion.

[0019] In another embodiment, the human milk-like product according to the invention can be adapted to the specific needs of the infant receiving it. The human milk-like product according to the invention can contain only one and / or some of the bioactive substances, macronutrients and micronutrients that can typically be found in human breast milk from a well-nourished mother. In such an embodiment, the human breast milk-like product may also be referred to by the term "non-standard human milk-like product". In one embodiment, a non-standard human milk-like product according to the invention comprises one or more nutrients or bioactives selected from the group consisting of proteins, peptides, lipids (including linoleic acid and alpha-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, myo-inositol, L-carnitine, growth / growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and / or exosomes), exosome-derived bioactives (e.g. miRNA) and secretory IgA.

[0020] In the context of the present invention, the term "unmodified human milk-like product" refers to the human milk-like product expressed by lactocytes and / or by the mammary gland-like organoids produced according to step A) and step B) of the method of the present invention, and not subjected to further processing according to optional step C) of the method of the present invention. Unmodified human milk-like product can include both standard and non-standard human milk-like products. Non-limiting examples of non-standard human milk-like products are selected from the group consisting of supplements, fortifiers, and raw materials enriched with only one and / or some of the bioactive substances, macronutrients, and micronutrients that can be typically found in human breast milk from well-nourished mothers.

[0021] In the context of the present invention, the term "modified human milk-like product" refers to a human milk-like product expressed by lactocytes and / or by mammary gland-like organoids generated according to steps A) and B) of the method of the present invention and subjected to further processing according to optional step C) of the method of the present invention.

[0022] Modified human milk-like products can include both standard and non-standard human milk-like products.

[0023] In the context of the present invention, the term "EB" means "embryoid body."

[0024] In the context of the present invention, the term "mEB" means "embryoid bodies cultured in MammoCult medium."

[0025] MammoCult Medium refers to a serum-free culture medium that contains basal medium, at least one growth supplement, heparin and hydrocortisone.

[0026] In the context of the present invention, the terms "embryoid bodies (EBs)", "embryoid bodies cultured in MammoCult medium (mEBs)", "mammospheres" and / or "spheroids" refer to the three-dimensional aggregates formed in suspension under step A) of the method of the present invention.

[0027] The term "infant" in the context of the present invention refers to a child under 12 months of age, such as under 9 months of age, in particular under 6 months of age.

[0028] In the context of the present invention, an infant may be either a full-term or a preterm infant. In one embodiment of the present invention, the infant is selected from the group of preterm and full-term infants.

[0029] The term "term infant" refers to an infant born at full term or at a gestational age of 37 weeks or greater.

[0030] The term "preterm infant" refers to an infant born at a gestational age less than 37 weeks.

[0031] In the context of the present invention, the term "birth weight" refers to the initial weight acquired by a fetus or newborn after birth.

[0032] In the context of the present invention, the term "low birth weight" means a birth weight below 2500 g (2499 g or less).

[0033] In the context of the present invention, the term "very low birth weight" means a birth weight of less than 1500 g (1499 g or less).

[0034] In the context of the present invention, the term "very low birth weight" means a birth weight of less than 1000 g (999 g or less).

[0035] The term "small-for-gestational-age infants" refers to infants whose birth weight is more than 2 standard deviations below the mean reference for birth weight on the gestational growth chart or 10 standard deviations below the mean reference for birth weight on the gestational growth chart. th Refers to infants who have a birth weight below the percentile. The term "small for gestational age" includes infants who are small at birth, either for constitutive or genetic reasons, or as a result of intrauterine growth restriction.

[0036] In the context of the present invention, the term "young children" or "toddlers" refers to children aged between 1 and 3 years.

[0037] The term "infant formula" as used herein refers to a nutritional composition intended for infants and as defined in Codex Alimentarius, (Codex STAN 72-1981) and foods for special dietary uses for infants (including foods for special medical purposes) as defined in Codex Alimentarius, (Codex STAN 72-1981). Infant formula also refers to a food intended for specific nutritional use in infants during the first month of life, which by itself meets the nutritional requirements of infants falling within this category (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant and follow-on formulas). Infant formula includes starter infant formulas and follow-up or follow-on formulas. Generally, starter formulas are breast milk substitutes for infants from birth, and follow-up or follow-on formulas are for those 6 months and older.

[0038] "Growing up milk" (or GUM) is given from the first year onwards. This milk is generally a milk-based drink that is specifically tailored to the nutritional needs of children. Such drinks are nutritional compositions that are used in combination with other foods to feed children from 12 months to 2-3 years of age.

[0039] In the context of the present invention, the term "fortifier" refers to a composition that contains one or more nutrients that have a nutritional benefit to an infant or young child.

[0040] The term "milk fortifier" refers to any composition used to fortify or supplement either human breast milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients. Thus, the human milk fortifier of the present invention can be administered after dissolution in human breast milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients, or it can be administered as a separate composition.

[0041] When administered as a separate composition, the human milk fortifier of the present invention may also be identified as a "supplement." In one embodiment, the human milk fortifier of the present invention is a supplement.

[0042] 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. A "human milk fortifier" according to the present invention may be intended to be administered to infants born prematurely with very low birth weight (VLBW) or extremely low birth weight (ELBW).

[0043] The milk fortifier according to the present invention may be a powder in liquid form.

[0044] Milk fortifier compositions having a liquid form offer some particular advantages: for example, liquid formulations may be more convenient when combined with packaging that delivers calibrated drops of a specific weight or volume.

[0045] Furthermore, liquid formulations are easier to mix with the composition to be fortified, while powder formulations may sometimes form lumps.

[0046] References herein to EpiCult Medium or EpiCultB Medium refer to serum-free culture medium containing hydrocortisone, insulin, FGF10 and HGF.

[0047] Culture media as disclosed herein refers to a solid, semi-solid or liquid containing essential nutrients designed to support the growth and differentiation of microorganisms. MammoCult medium is one example of a culture medium that may be used in the present invention.

[0048] Methods and uses according to the present invention The present invention relates to a method for producing mammary gland cells using mammalian epithelial cells cultured under specific conditions, and to a method for producing a mammalian milk-like product in vitro using said mammary gland cells.

[0049] Surprisingly, the present invention has demonstrated that mammary epithelial cells can be used as starting material in the protocol for producing mammalian milk-like products.The use of mammary epithelial cells as starting material means that, for example, there is no need for a complicated differentiation protocol for first producing mammary epithelial cells from early-stage undifferentiated stem cells.This elimination of the need has the advantage of shortening the overall time and saving costs of the method for producing mammalian milk-like products compared to stem cell protocols.

[0050] Accordingly, the present invention provides a method for producing a mammalian milk-like product, comprising the steps of: A) culturing mammary epithelial cells in a culture medium to generate lactocytic mammary-like gland organoids; B) secreting a mammalian milk-like product from said lactocytes.

[0051] Production of mammalian milk-like products The present invention relates to a method for producing a mammalian milk-like product as defined herein, comprising any of steps A) and B) as defined herein, and an optional step C) as defined herein.

[0052] Step A. Generating lactocytes and / or mammary-like organoids According to the method of the present invention, mammary gland-like cells and / or organoid structures are generated under step A).

[0053] This generation involves the growth and maturation of mammary epithelial cells to produce mammary-like organoids (e.g., lactocytes).

[0054] In the methods disclosed herein, mammary epithelial cells are used as the starting material, i.e., the culture of the epithelial cells is the first step of the method.

[0055] The growth and maturation of mammary epithelial cells occurs by culturing them in a specific culture medium, for example complete MammoCult medium (StemCell Technologies). Complete MammoCult medium is preferably composed of basal medium, growth supplements, heparin (typically 4 μg / mL), and hydrocortisone (typically 0.48 μg / mL). The medium is usually changed every 3 days. The mEBs (mammospheres) obtained in the previous step are then enriched with non-neurectodermal cells.

[0056] In some embodiments, the growth and maturation stage is from day 0 to day 7, where day 0 is the time when the mammary epithelial cells are first added to the culture medium. In some embodiments, the growth and maturation stage is 7 days. In some embodiments, the growth and maturation stage is 7 days or less.

[0057] After the growth and maturation stage, the cells are induced to express milk proteins. In some embodiments, the induction period is between 7 and 14 days. In some embodiments, the induction period is 7 days. In some embodiments, the induction period is 7 days or less.

[0058] In one embodiment of the present invention, a method for producing a human milk-like product is provided, comprising generating lactocytes from human mammary epithelial cells under step A), said step A) comprising: A method is provided which comprises: i) culturing human mammary epithelial cells in an appropriate culture medium (eg, MammoCult medium) to generate lactocytes after 7 days.

[0059] In another embodiment, a method for producing a human milk-like product is provided, comprising generating lactocytes from human mammary epithelial cells under step A), said step A comprising: i) culturing human mammary epithelial cells under non-adherent conditions in an appropriate culture medium (eg, MammoCult medium) for at least 7 days to generate lactocytes.

[0060] In one embodiment, the method according to the invention provides culture conditions according to step A) that are adjusted to generate lactocytes derived from human mammary epithelial cells capable of secreting a human milk-like product.

[0061] In preferred embodiments, there is provided a method for producing a human milk substitute product, comprising generating lactocytes from human mammary epithelial cells under step A), said step A) comprising growing and maturing the mammary epithelial cells to differentiate into mammary cells (e.g., lactocytes) in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) as described herein. In some embodiments, for at least 7 days. In some embodiments, for 7 days or less.

[0062] In another preferred embodiment, a method for producing a human milk-like product is provided, comprising generating lactocytes from human mammary epithelial cells under step A), said step A) comprising: i) culturing mammary epithelial cells in a suitable culture medium (e.g., MammoCult medium) in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) for at least 7 days (days 0 to 7) to generate lactocytes.

[0063] In a particularly preferred embodiment of the present invention, a method for producing a human milk-like product is provided, which comprises generating lactocytes from human mammary epithelial cells under step A), wherein: i) culturing mammary epithelial cells for 7 days (days 0-7) in complete MammoCult medium (StemCell Technologies) containing basal medium, growth supplements, and supplemented with heparin (typically 4 μg / mL), hydrocortisone (typically 0.48 μg / mL); and ii) inducing expression of the milk protein by incubating the cells for 7 days (7-14 days) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0064] Step ii) preferably results in differentiation into milk protein expressing cells, in particular lactocytes, and / or mammary gland-like organoids.

[0065] In a further particularly preferred embodiment of the present invention, a method for producing a human milk-like product is provided, which comprises generating lactocytes from human mammary epithelial cells under step A), wherein i) culturing mammary epithelial cells as described herein in MammoCultB medium supplemented with MammoCult growth supplement, hydrocortisone, and heparin for 7 days (days 0 to 7); and ii) inducing expression of the milk protein by incubating the cells for 7 days (7-14 days) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0066] Step ii) preferably results in differentiation into milk protein expressing cells, in particular lactocytes, and / or mammary gland-like organoids.

[0067] In one embodiment, step ii) as defined above for the particularly preferred embodiment preferably results in the formation / differentiation of at least breast cells, luminal cells and basal cells. In this context, breast cells preferably express one or more, preferably all, of the markers selected from the group consisting of β-casein, milk proteins and hormone receptors. Furthermore, luminal cells preferably express one or more, preferably all, of the markers selected from the group consisting of EpCAM, MUC1, CD49F, GATA3, CK8 and CK18. Furthermore, basal cells preferably express one or more markers selected from the group consisting of CK14, α-smooth muscle actin and P63.

[0068] In a further embodiment, after step ii) as defined above for the particularly preferred embodiment, mammary-like organoids can be obtained which express one or more markers selected from the group consisting of β-casein, milk proteins and hormone receptors, luminal cells which express one or more markers selected from the group consisting of EpCAM, MUC1, CD49F, GATA3, CK8, CK18, and basal cells which express one or more markers selected from the group consisting of CK14, α-smooth muscle actin and P63.

[0069] In one embodiment of the present invention, the above method is provided for producing a human milk-like product.

[0070] In one embodiment (of step A), delivery of nutrients and biomimetic stimuli is controlled to affect cell growth, differentiation, and tissue formation. In one embodiment (of step A), such control is performed within a bioreactor.

[0071] In some embodiments, the mammary gland-like organoids or lactocytes derived from step A have high expression of mammary gland-specific markers. In some embodiments, the mammary gland-like organoids or lactocytes derived from step A express keratin 18 (KRT-18). In some embodiments, the mammary gland-like organoids or lactocytes derived from step A express estrogen receptor (ER). In some embodiments, the mammary gland-like organoids or lactocytes derived from step A express more than 59% KRT-18 and ER as estrogen receptor positive luminal mammary gland population.

[0072] In some embodiments, the mammary gland-like gland organoids or lactocytes derived from step A have high expression of important milk bioactive substances. In some embodiments, the mammary gland-like gland organoids or lactocytes derived from step A have increased expression of important milk bioactive substances. In some embodiments, the mammary gland-like gland organoids or lactocytes derived from step A have increased expression of important milk bioactive substances after induction (e.g., day 14) compared to before induction (e.g., day 7). In some embodiments, the mammary gland-like gland organoids or lactocytes derived from step A have increased mRNA expression of lactoferrin (LTF) after induction compared to before induction. In some embodiments, the mammary gland-like gland organoids or lactocytes derived from step A have increased mRNA expression of MFGE8 (milk fat globule-EGF factor 8) after induction compared to before induction.

[0073] It will be appreciated that any of the methods or method steps disclosed herein may be performed in three-dimensional suspension culture rather than using a membrane matrix as a support. Thus, in some embodiments, cells are maintained in suspension culture during the entire differentiation procedure.

[0074] Step B. Expression of human breast milk-like products In one embodiment of the present invention, the method comprises expressing human milk-like products from mammary gland-like organoids derived from human mammary epithelial cells, preferably prepared according to step A. The expression of human milk-like products preferably occurs upon induction of the expression of human milk-like products from such lactocytes and / or mammary gland-like organoids.

[0075] In one embodiment, lactating lactocytes are induced by application of a specific medium (eg, EpiCultB) supplemented with lactogenic factors (eg, prolactin, hydrocortisone, and insulin).

[0076] In particular, the human milk-like product obtained from mammary-like organoids derived from human mammary epithelial cells, preferably prepared according to step A), contains human milk bioactive substances selected from the group consisting of or including proteins, lipids or oligosaccharides, preferably human milk oligosaccharides, etc. Using the particularly preferred protocol according to steps A i) to iv) implemented above, the inventors have succeeded in identifying, inter alia, oligosaccharides (including lactose and some HMOs), lipids (including 4 fatty acids), proteins (7 detected including casein), and miRNAs (75 detected, including 11 typically detected in HBM).

[0077] In one embodiment, the human milk-like product obtained from mammary-like organoids derived from human mammary epithelial cells, preferably prepared according to step A), contains human milk bioactive substances, including or selected from the group consisting of oligosaccharides, lipids, proteins, exosomes and miRNA.

[0078] In another embodiment, the human milk-like product obtained from mammary-like organoids derived from human mammary epithelial cells, preferably prepared according to step A), is preferably selected from the group consisting of lactose, 6'SL, C-4:0 fatty acid, C-8:0 fatty acid, C-10:0 fatty acid, C-14:0 fatty acid, C-15:0 fatty acid, C-16:0 fatty acid, C-16:1n7 fatty acid, C-17:0 fatty acid, C-18:0 fatty acid, C-18:1 n9 fatty acid, C-18:1 fatty acid, C-18:2 n6 fatty acid, C-20:0 fatty acid, C-20:1 n9 fatty acid, C-18:3 The human milk bioactives include or are selected from the group consisting of n3 fatty acids, C-22:0 fatty acids, lactoferrin, albumin, prolactin, alpha S1-casein, hemoglobin subunit beta, hemoglobin subunit alpha, α-lactalbumin, alpha-2-macroglobulin, β-casein, bile salt activated lipase, κ-casein, lactadherin, 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.

[0079] In one embodiment of the present invention, the human milk-like product obtained from the mammary gland-like organoid derived from human mammary epithelial cells is standard human milk product.In another embodiment of the present invention, the human milk-like product obtained from the mammary gland-like organoid derived from human mammary epithelial cells is non-standard human milk product.

[0080] Step C. Further processing to produce a modified human breast milk-like product In one optional embodiment of the invention, the method described herein comprises an additional step C) performed on the human milk-like product obtainable from step B) which comprises subjecting such product to further processing to provide a modified human milk-like product.

[0081] In a particular embodiment, the additional processing step C) performed on the human breast milk-like product of the present invention may be selected from the group consisting of a purification step, an isolation process, an extraction process, a fractionation step, an enrichment process, an enzymatic treatment, the addition of further components (e.g. those that cannot be expressed by human mammary organoids, such as, for example, immunoglobulins, probiotics and / or minerals) or a combination thereof.

[0082] Human Milk-Like Products "Standard" human milk-like product In one embodiment of the invention, the human breast milk-like product is a "standard" human breast milk-like product, i.e. it contains the same components as human breast milk from well-nourished mothers.

[0083] The benefits of breastfeeding are well known in the scientific literature and having access to human breast milk-like products would allow such products to be used for many equally well-known health benefits.

[0084] In such embodiments, the human breast-milk-like product can be used as a breast-milk substitute in situations where actual breast-feeding is not possible.

[0085] In such embodiments, the human breast milk-like product is intended to be used, for example, to support a longer breastfeeding experience in women who have low lactation or who have stopped lactating six months after giving birth.

[0086] Similarly, human breast milk-like products are intended to be used to enable lactation even in situations where, for example, illness impairs actual lactation from the mother.

[0087] In another embodiment, the human breast milk-like product is intended for use in situations where breast milk production does not begin naturally, such as when an infant is adopted.

[0088] In one embodiment, the human milk-like product according to the present invention is not a product of human breast milk lactation as it occurs in nature.

[0089] In one embodiment, the human breast milk-like product is for use in providing optimal nutrition to infants.

[0090] In one embodiment, the human breast milk-like product is for use in providing healthy development to an infant.

[0091] In one embodiment, the human breast milk-like product is for use in preventing infection, obesity and promoting immune development in young children.

[0092] In one embodiment, the human breast milk-like product is an unmodified human breast milk-like product.

[0093] In another embodiment, the human breast milk-like product is a modified human breast milk-like product.

[0094] In one embodiment, the human milk-like product according to the present invention comprises proteins, lipids, carbohydrates, vitamins and minerals.

[0095] In another embodiment, the human milk-like product according to the present invention comprises proteins, lipids, carbohydrates, vitamins, minerals and bioactive substances.

[0096] 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, myo-inositol and L-carnitine.

[0097] In a further embodiment, the human milk-like product according to the invention also comprises at least one biologically active substance selected in the group consisting of proliferation / growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and / or exosomes), exosome-derived biologically active substances (e.g. miRNA) and secretory IgA.

[0098] Such human breast milk-like products may be prepared according to the methods of the present invention, for example, by including step C) of adding proliferation / growth factors, cytokines, probiotics, extracellular vesicles (e.g., milk fat globules and / or exosomes), exosome-derived bioactive substances (e.g., miRNA) and secretory IgA.

[0099] In one embodiment the human breast milk-like product contains probiotics.

[0100] Such human breast milk-like products can be prepared according to the methods of the present invention, for example, by including probiotics (e.g., B. Lactis, B. Infantis, L. Ramnhosus), which can be obtained from a number of commercially available sources.

[0101] In such embodiments, the human breast milk-like product may be used to optimize gastrointestinal function and / or promote immunity.

[0102] In one embodiment, the human breast milk-like product contains secretory IgA and probiotics.

[0103] Such a human breast milk-like product may be prepared according to the methods of the invention by including step C) of adding a combination of probiotics and secretory IgA, which may be prepared as described, for example, in WO 2009 / 156301 and WO 2009 / 156367, which are incorporated herein by reference. In such an embodiment, the human breast milk-like product may be used to prevent immunoglobulin deficiencies and / or in the prevention of recurrent infections in infants and young children.

[0104] "Non-standard" human milk-like products In one embodiment of the present invention, the human milk-like product may be altered from the component ratios and concentrations naturally found in human breast milk from well-nourished mothers, referred to herein as a "non-standard milk-like product."

[0105] In one embodiment, the human milk-like product according to the present invention may be selected from the group consisting of milk fortifiers, supplements, and / or human breast milk substitutes adapted for special purposes.

[0106] Human Milk Fortifiers and Human Milk Bioactive Supplements In one embodiment, the method of the present invention provides a human breast milk-like product that can be used to fortify human breast milk obtained naturally from lactating mothers or to fortify infant formula.

[0107] In another embodiment, the method of the present invention provides a human breast milk-like product that may be used as a supplement for infants or young children in need thereof.

[0108] In such embodiments, the human breast-milk-like products may be used to provide healthy development and / or reduce the risk of developing illnesses typically associated with certain conditions in infants or young children (e.g., asthma, allergies, cognitive changes, etc.) and / or promote growth, immune development, and protection from infection.

[0109] Notably, the human origin of the ingredients (especially the bioactive ingredients) in such fortifiers or supplements, combined with the fact that they are produced by the methods of the present invention, is believed to provide such ingredients with intact or enhanced functionality.

[0110] The human breast milk-like product is preferably intended for use as a fortifier. Such a human breast milk-like product is intended for use as a fortifier and can for example be prepared according to the method of the invention by including step C) of isolation and / or enrichment of (certain) bioactive substances from the human breast milk-like product obtainable from step B). Such an isolation step may be carried out via classical fractionation, enrichment and / or purification of the unmodified human breast milk-like product obtainable from step B).

[0111] Human breast milk-like products intended for use as supplements may include one or more bioactive substances selected from the group consisting of human milk oligosaccharides (e.g., 2 FL, 3 FL, LNT, LnNT, DiFl, 6SL and / or 3SL), lipids, growth / growth factors (e.g., epidermal growth factor (EGF), heparin-binding epidermal growth factor), cytokines (e.g., transforming growth factor-β2 (TGFβ-2), IL-1, IL-2, IL-6, IL-10, IL-18, interferon gamma (INF-γ), TNF-α), extracellular vesicles (e.g., milk fat globules and / or exosomes), exosomes containing microRNA, and antimicrobial / protective bioactive substances (e.g., IgA, lactoferrin, lysozyme, lactadherin). Such a human breast milk-like product intended for use as a supplement may for example be prepared according to the method of the invention by including step C) of isolating biologically active substances from the unmodified human breast milk-like product obtainable from step B. Such an isolation step may be carried out via classical fractionation, enrichment and / or purification of the unmodified human breast milk-like product obtainable from step B).

[0112] 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, for use in fulfilling the profile of human breast milk from women who do not secrete fucosylated oligosaccharides due to an inactive FUT2 gene.

[0113] Such a human breast milk-like product intended for use as a fortifier or supplement may be prepared according to the method of the invention, for example by including step C) of isolating and / or enriching fucosylated oligosaccharides (e.g. 2FL and / or 3FL) from the unmodified human breast milk-like product obtainable from step B).

[0114] In such embodiments, the human breast milk-like product may be used to optimize gastrointestinal function and / or promote immunity.

[0115] Human breast milk-like products for infants with genetic disorders In one embodiment, the human breast milk-like product according to the present invention may be adapted to address the specific needs of infants born with genetic disorders.

[0116] Galactosemia In such embodiments, the human breast milk-like product can be adapted to the needs of infants suffering from galactosemia, a rare genetic disorder that affects an infant's ability to metabolize galactose.

[0117] In such embodiments, the human breast milk-like product should be depleted of lactose and / or lactose containing saccharides, hi such embodiments, the human breast milk-like product may be used to provide healthy development for infants with galactosemia.

[0118] In one embodiment, a human breast milk-like product from which lactose and / or lactose-containing saccharides have been removed may be obtained according to the method of the invention by including a step C) of enzymatic treatment (lactase treatment) or a step C) of membrane fractionation and ultrafiltration of the unmodified human breast milk-like product obtained from step B).

[0119] Phenylketonuria In such an embodiment, the human breast milk-like product can be adapted to the needs of infants suffering from phenylketonuria (PKU), which is caused by the absence or dysfunction of phenylalanine hydroxylase, which converts phenylalanine to tyrosine. If untreated, brain toxicity leads to severe mental retardation.

[0120] In such embodiments, the human breast milk-like product should be deprived or depleted of phenylalanine.

[0121] In such embodiments, the human breast milk-like product may be used to provide healthy development for infants affected by PKU.

[0122] In one embodiment, the human breast milk-like product is depleted in phenylalanine such that the phenylalanine content is maintained below 20 mg / kg body weight of the subject receiving it.

[0123] In one embodiment, a phenylalanine-depleted or removed human breast milk-like product can be obtained according to the method of the invention by including a step C) of enzymatic treatment (proteolysis) or filtration of the unmodified human breast milk-like product obtained from step B).

[0124] In one embodiment, a phenylalanine-depleted human breast milk-like product can be obtained according to the method of the invention by including a step C) of enzymatic treatment (proteolysis) or filtration of the unmodified human breast milk-like product obtained from step B).

[0125] In another embodiment, a phenylalanine-depleted human breast milk-like product can be obtained according to the method of the present invention by providing in step B) a culture medium that provides limited or zero amounts of phenylalanine, such as a culture medium containing whey-derived glycomacropeptide (GMP).

[0126] Additional Embodiments of the Invention The following is provided:

[0127] S1. A method for producing a mammalian milk-like product, comprising: A) culturing mammary epithelial cells in a culture medium to generate lactocytic mammary-like gland organoids; B) secreting said mammalian milk-like product from said lactocytes.

[0128] S2. The method according to statement 2, wherein the duration of step A) is less than or equal to 14 days, optionally 14 days.

[0129] S3. The method according to statement 1 or 2, wherein the lactocytic mammary-like gland organoids from step A) express one or more mammary positive cell markers, optionally selected from KRT-18 and ER.

[0130] S4. The method according to any one of statements 1 to 3, wherein the lactocytic mammary-like gland organoids from step A) have increased mRNA expression of one or more milk bioactivity markers after induction compared to before induction.

[0131] S5. The method according to any one of statements 1 to 4, wherein the lactocytic mammary-like gland organoids from step A) have increased LTF mRNA expression after induction compared to before induction.

[0132] S6. The method according to any one of statements 1 to 5, wherein the lactocytic mammary-like gland organoids from step A) have increased MFGE8 mRNA expression after induction compared to before induction.

[0133] S7. The method according to any one of statements 1 to 6, wherein the culture medium is MammoCult medium in a suitable three-dimensional culture system, e.g., in three-dimensional suspension conditions.

[0134] S8. Step A) further comprises: i) culturing said mammary epithelial cells; and ii) inducing milk protein expression.

[0135] S9. The method according to statement 8, wherein i) of step A is for no more than 7 days, optionally for 7 days.

[0136] S10. The method according to statement 8 or 9, wherein step Aii) is for no more than 7 days, optionally for 7 days.

[0137] S11. Step A) i) culturing the mammary epithelial cells for 7 days in complete MammoCult medium containing basal medium, growth supplements, and supplemented with heparin and hydrocortisone; ii) inducing milk protein expression by incubating the cells in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days.

[0138] S12. Step A) i) culturing the mammary epithelial cells for 7 days in MammoCultB medium supplemented with MammoCult growth supplement, hydrocortisone and heparin; ii) inducing milk protein expression by incubating the cells in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days.

[0139] S13. The method of any one of statements 1-12, optionally including step C) of further processing the milk-like product to produce a conditioned mammalian milk-like product.

[0140] S14. The method according to any one of statements 1 to 13, wherein the mammary epithelial cells are human mammary epithelial cells.

[0141] S15. A human milk-like product obtainable according to the method of statement 14.

[0142] S16. A human milk-like product as described in statement 15 for use in therapy.

[0143] S17. Use of a human milk-like product as described in statement 15 as a human milk substitute, optionally as a breast milk substitute.

[0144] It should be understood that the various aspects and embodiments of the detailed description as disclosed herein are illustrative of specific ways to make and use the invention, and are not intended to limit the scope of the invention when considered in conjunction with the claims and the detailed description. It should also be understood that features from aspects and embodiments of the invention may be combined with additional features from the same or different aspects and embodiments of the invention.

[0145] As used in the detailed description and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. [Brief description of the drawings]

[0146] [Figure 1] FIG. 1 shows differentiation of human induced pluripotent stem cells (hiPSCs) according to the protocol outlined in Ying Qu and applied to one alternative method in step A) of the method of the present invention. [Diagram 2] Step A) Differentiation of human induced pluripotent stem cells (hiPSCs). [Diagram 3] Figure 2 shows that 3D organotypic cultures of hiPSCs produced according to the method are highly permissive for mammary specification. The mRNA markers of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8 and CSN2 in the 3D differentiation (42 days) protocol indicate the stages of pluripotency (Nanog), lineage (ectoderm and endoderm) (TUBB3, FOXA2), basal cell / myoepithelial markers (TP63, KR-14), luminal epithelial markers (EpCAM, KRT8), and milk protein (CSN2 (casein beta)). [Figure 4] 2D organotypic cultures of hiPSCs produced as comparative examples are shown. mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8, and CSN2 is shown during the 2D differentiation (31 day) protocol. Markers indicate stages from left to right: pluripotency (Nanog), lineage (ectoderm and endoderm) (TUBB3, FOXA2), basal cell / myoepithelial markers (TP63, KR-14), luminal epithelial markers (EpCAM, KRT8), and milk protein (CSN2 (casein beta)). [Diagram 5]Figure 1 shows 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 (D7-14). b, Flow cytometric analysis of mammary markers keratin 18 and estrogen receptor during the growth phase of mammary epithelial cell cultures. c, RNA expression (ΔCt) levels of human lactoferrin (LTF) and milk fat globule-EGF factor 8 (MFGE8) / lactadherin during the growth phase (day 7) and lactation induction phase (day 14). The ΔCt method was used for panel presentation, and LTF and MFGE8 genes are ranked by calculating the mean standard deviation (SD) based on the relative expression of the candidate reference genes. Genes with the lowest SD among the listed genes were identified as the most stable or most expressed genes. [Figure 6] Using NanoString technology for gene expression profiling, we show the expression of various mammary epithelial markers in mammary epithelial cells (a–h).

[0147] experiment Example 1 Obtaining human milk-like products by culturing hiPSCs and differentiating them into lactocytes Ying Qu et al,Stem Cell Report vol 8,205-215 February 14 th Starting from ihPSCs, lactocytes can be cultured according to the procedure described in 2017, and the human milk-like products secreted thereby can be recovered and used in the therapy according to the invention and / or as breast milk substitute.

[0148] Example 2 Obtaining a human milk-like product by culturing hiPSCs and differentiating them into 3D-lactocytes Starting from hiPSCs according to the method of the invention, lactocytes are cultured according to steps A) and B) above and the human milk-like product secreted thereby can be recovered and used in therapy and / or as a breast milk substitute according to the invention.

[0149] Example 3 Obtaining a human milk-like product by an alternative method of culturing hiPSCs and differentiating them into lactocytes Efficient lactocyte differentiation from hiPSCs can be obtained from alternative culture conditions, including conditions 1 to 4 described below. 1. EB-derived cells are cultured as monolayers on vitronectin-coated plates in 2D culture for at least 28 days in medium containing RPMI 1640 supplemented with L-glutamine, fetal bovine serum (FBS), insulin, epidermal growth factor (EGF), hydrocortisone, and Pen-Strep penicillin / streptomycin: antibiotic-antimycotic solution. 2. Two-dimensional culture of adherent aggregates of cells derived from EBs (EB) on vitronectin-coated plates in medium containing RPMI 1640 supplemented with L-glutamine, fetal bovine serum (FBS), insulin, epidermal growth factor (EGF), hydrocortisone, and Pen-Strep (antibiotic-antimycotic solution) for at least 28 days. 3. 3D culture in suspension in MammoCult medium for at least 10 days, then culture in specific medium (e.g., EpiCultB) in the presence of parathyroid hormone for an additional 5 days, followed by culture in a mixed floating gel (e.g., Matrigel and Collagen 1) in the presence of insulin, HGF, hydrocortisone and FGF10 for 25 days. 4. EBs are cultured in 3D in suspension in MammoCult medium (ultra-low attachment plates) for at least 10 days, then in specific medium (e.g. EpiCultB) in the presence of parathyroid hormone for an additional 5 days, followed by 25 days in the presence of insulin, HGF, hydrocortisone and FGF10.

[0150] Example 4 Two- and three-dimensional differentiation of human induced pluripotent stem cell (hiPSC) line 603-based lactocytes (a) Three-dimensional differentiation of human induced pluripotent stem cell (hiPSC) line 603-based lactocytes: For three-dimensional differentiation of lactocytes, we used human induced pluripotent stem cell (hiPSC) line 603. Human induced pluripotent stem cell (hiPSC) line 603 was purchased from Fujifilm Cellular Dynamics, Inc. (FCDI). (i) For the three-dimensional differentiation protocol (according to the present invention), single cells of hiPSCs were formed into EBs (spheroids) by incubating overnight in E8 medium containing 10 uM rock inhibitor at 37° C., 5% CO2, and rotating at 95 rpm. On day 2, the medium was replaced with E8 (day -2 to day 0). The next day, the medium was replaced with Mammo1 medium (MammoCult medium supplemented with penicillin / streptomycin and growth supplements, heparin (4 μg / mL), and hydrocortisone (0.48 μg / mL)) for 10 days (day 0-10). Medium was replaced every 2 days. (ii) After differentiation, the cells were cultured in Mammo2 medium (EpiCultB + supplements, PTHrP 100 ng / ml + penicillin / streptomycin) for 5 days. The culture medium was changed every 3 days (days 10 to 15). (iii) To induce branching of epithelial structures, alveolar differentiation and mammary specification, mEBs (spheroids / mammospheres) 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 (days 15-35). (iv) Finally, to induce milk bioactive production (3D), Mammo4 medium (complete EpiCultB, 10% FBS, prolactin (10 μg / ml), hydrocortisone (1 μg / ml), insulin (10 μg / ml), progesterone, β-estradiol and penicillin / streptomycin) was used for 7 days, with the medium changed every 3 days (days 35-42). During all differentiation procedures, spheroids were maintained in suspension culture (rotating at 95 rpm). The differentiation procedure was terminated on day 42. The results are represented in Figure 3.

[0151] (b) Two-dimensional differentiation of human induced pluripotent stem cell (hiPSC) line 603-based lactocytes: For two-dimensional differentiation of lactocytes, we also used human induced pluripotent stem cell (hiPSC) line 603. Human induced pluripotent stem cell (hiPSC) line 603 was purchased from Fujifilm Cellular Dynamics, Inc. (FCDI). In the 2D differentiation protocol (used for comparison), we used Lacto medium (RPMI 1640, 20% FBS, 1 mM glutamine, 4 μg / ml insulin, 20 ng / ml EGF, 0.5 μg / ml hydrocortisone, penicillin / streptomycin) during all differentiation steps. Cells were incubated at 37° C., 5% CO2. Medium was changed every 2 days. The results are presented in FIG. 4.

[0152] (c) Results Quantitative RT-PCR was used to capture each stage of differentiation during lactocyte induction (Figure 3, 3D differentiation; Figure 4, 2D differentiation). In both 2D and 3D settings, NanoG expression as a marker of pluripotency decreases as cells mature and differentiate. Neuroectodermal and endodermal markers, TUBB3 (Tubulin beta 3 class III) and Forkhead box protein A2 (FOXA2), are not significantly expressed in 3D format, and elevated TUBB3 is only captured in 2D settings. This demonstrates that hiPSCs are patterned towards non-neurectodermal lineages and thus enriched for mammary progenitors in 3D format. We tested the expression patterns of commonly used basal cell / myoepithelial markers such as p63 (nuclear protein with homology to p53) and cytokeratin 14 (KRT-14). Both markers are significantly detectable in both systems. Furthermore, epithelial cell adhesion molecule (EpCAM) and cytokeratin 8 (KRT8) were tracked only in the three-dimensional system, while KRT8 was only partially expressed in the two-dimensional format. As a result, the three-dimensional plates in the organ-type setting expressed both breast tissue markers, luminal markers, and basal markers. Such mammary-like organoids express human breast-specific proteins, including CSN2 (casein beta), milk protein peptides, and hormone receptors. Luminal cells specifically express EpCAM, MUC1, CD49F, GATA3, CK8, and CK18, whereas basal cells specifically express CK14, α-smooth muscle actin, and P63. Finally, EpCAM and CD49F double positive cells can be detected at the early progenitor stage between D10 and D35. Interestingly, CSN2 expression is only captured at the end time point (D42) in the three-dimensional organ system, but not in the two-dimensional differentiation platform.

[0153] Analysis of the secretome of mammary-like organoids demonstrated secretion of human milk-specific bioactive substances including oligosaccharides (including lactose and several HMOs), lipids (including four fatty acids), proteins (seven detected including casein), and miRNAs (75 detected including 11 typically detected in HBM), as described below.

[0154] The supernatants of primary cells were analyzed for the presence of lactose or human milk oligosaccharides according to the procedure described in Austin and Benet, Quantitative determination of non-lactose milk oligosaccharides, Analytica Chimica Acta 2018, 1010, 86-96, with minor modifications. Samples were analyzed by UHPLC and detected lactose or human milk oligosaccharides (HMOs) were quantified against a calibration curve of lactose and a mixture of seven HMOs (2'FL, 3'FL, DFL, LNT, LNnT, 3'SL and 6'SL). The method was estimated to have a limit of 0.1 mg / L. In the supernatants of primary cells, lactose (0.22 mg / L) and 6'SL (0.32 mg / L) were detected at day 42.

[0155] The fatty acids in the medium and cell supernatants were analyzed by gas chromatography coupled with a flame ionization detector. Briefly, the supernatants obtained on day 42 are analyzed to investigate the presence of fatty acids in several lipid classes. A 7890A gas chromatograph equipped with a 7693 autosampler with a preparative station module equipped with a fused silica CP-Sil 88 capillary column (100% cyanopropylpolysiloxane, 100 m, 0.25 mm inner diameter, 0.25 mm film thickness) is used with a split injector (1:25 ratio) heated at 250 °C, and a flame ionization detector operated at 300 °C. The preparation of FAMEs (fatty acid methyl esters) is carried out by direct transesterification of the samples with methanolic chloridric acid. The separation of the FAMEs is carried out using capillary gas chromatography-FID (GC). The identification of the FAMEs is carried out by retention time (RT) and comparison with external standards. Quantification of fatty acids is performed by calculation using methyl C11:0 as internal standard. The transesterification performance of the method is controlled using TAG C13:0 as a second internal standard. After addition of the internal standard, the solution was mixed with 2 mL of methanol, 2 mL of methanol / HCl (3N) and 1 mL of hexane. After heating at 100°C / 60 min, the sample was cooled to room temperature (approximately 15 min) and the reaction was stopped by adding 2 mL of water. After centrifugation, the organic phase was directly injected into the GC.

[0156] The fatty acid results (differences observed between medium and supernatant) from the protocol of Example 4a at day 42 are shown in Table 1.

[0157] Table 1 below lists the fatty acids expressed in the cell supernatant samples.

[0158] [Table 1]

[0159] Proteins in cell supernatants were analyzed using SDS-PAGE profiling followed by band isolation for identity confirmation by LC-MSMS. For SDS-PAGE analysis, the entire amount of prepared sample was loaded onto the gel. Human milk sample was added as a control for comparison. Selected gel regions (bands) were cut to explore human proteins by LC-MSMS. Finally, the bands were subjected to in-gel trypsin digestion and analyzed by LC-MSMS. The LC-MSMS data was analyzed in Peaks Studio and matched against the UniProt database of human proteins.

[0160] Table 2 below lists the best candidates for all excised bands.

[0161] [Table 2]

[0162] Exosome isolation and miRNA profiling were performed using ExoQuick polymer nets. The ExoQuick polymer acts to precipitate exosomes by forming a network, recovering all exosomes of a certain size. Once the ExoQuick mesh is formed, the exosomes are easily pelleted by simple low-speed centrifugation. The exosomes are intact, ready for protein or RNA analysis, and bioactive for functional testing. Precipitation buffer was added to the samples at a ratio of 0.25x and then vortexed. The mixture was incubated overnight at 4°C. After incubation, the samples were centrifuged at 1500xg for 30 min. The exosome pellet was resuspended by vortexing in the initial volume with Buffer XE (QIAGEN) for QC or Lysis Buffer from HTG EdgeSeq miRNA Whole Transcriptome Assay for miRNA profiling. To evaluate the isolation of extracellular vesicles (EVs), the supernatant was first centrifuged at 3000g for 15 min to remove cell pellets and debris. Then, 100 microliters of medium was used to precipitate with ExoQuick buffer (0.25x ratio) overnight at 4°C. The EV precipitate was collected by centrifugation at 1500g for 30 min. Two precipitations were performed for each sample, the first EV precipitate was resuspended in buffer XE (QIAGEN) for possible further analysis, and the second precipitate was resuspended in 50 ul of HTG lysis buffer only for 10-fold concentration, and then subjected to miRNA profiling by HTG.

[0163] For miRNA profiling, samples were used directly in the first lysis step. Thus, the entire sample was used directly and lysed in a 1:1 ratio with plasma lysis buffer. Proteinase K (1 / 10) was then added and the sample was incubated for 3 h at 50 °C and 600 rpm on a Thermomixer. EVs were resuspended in lysis buffer and lysed in the same conditions, with an additional incubation step of 10 min at 95 °C before the lysis incubation. 26 μL of lysate was processed with 70 μL of oil on an HTG processor according to the HTG EdgeSeq miRNA Whole Transcriptome Assay V2 procedure. For indexing and amplified libraries, samples were tagged with Illumina adapters and PCR-run with OneTaq® Hot Start 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) and AMPure washed (2.5 ratio) on a robotic liquid handler SciClone NGS WorkStation (Perkin Elmer). Pools were obtained using our custom pooling program on a Hamilton robot. Samples were pooled based on GX Touch Tip HS quantification. In a second run, pools were manually purified with AMPure Beads (1.8 ratio) to remove possible remaining traces of primer dimers, quantified using Qubit, and the final concentration was adjusted to 2 nM. As a final step, for MiSeq sequencing, 20 pM of the pool was loaded onto the MiSeq with 5% PhiX added and sequenced on the MiSeq for 50 base single reads using the 150V3 kit.

[0164] Briefly, 974 miRNAs were detected in cell supernatants, of which more than 75 were highly expressed miRNAs in milk samples.

[0165] Table 3 below lists the top 10 highly expressed miRNAs.

[0166] [Table 3]

[0167] Our findings provide a novel iPSC-based three-dimensional organotypic model to study the regulation and development of normal mammary cell fate and function, and breast milk bioactive production.

[0168] Example 5 Mammary epithelial cells were cultured in a three-dimensional format with and without matrix for a 7-day growth phase and a 7-day induction phase using differentiation medium M1 / M4 (Figure 5a). Other different media may be used. Briefly, 1-5 million dissociated epithelial cells were seeded in 6-well plates (ultra-low attachment) containing 4.5 mL of medium using a planar shaker platform. The cultured cells can express mammary specific markers such as keratin 18 and estrogen receptors (Figure 5b) and mRNA levels of human lactoferrin (LTF) and milk fat globule-EGF factor 8 (MFGE8) or lactadherin during the growth phase (day 7) and induction phase (day 14) using flow cytometric quantification (Figure 5c, d).

[0169] We used NanoString technology for gene expression profiling to assess the expression of various breast epithelial markers in mammary epithelial cells (Figure 6). The mammary epithelial progenitor cell marker CD24 was consistently expressed and tended to be highly expressed during lactation (Figure 6a). It is interesting to observe that as lactation progresses, the cells show a decrease in expression of markers of mammary basal-like cells such as KRT5, KRT14 and ITGA6 (CD49f) (Figure 6b-d). The cells appear to have maintained a luminal-like phenotype during and after lactation using various markers such as EpCAM, KRT8, KRT18 and MUC1 (Figure 6e-h). The post-induction period is assessed by the onset of lactocyte (luminal-like cell) specific secretory profiles for different proteins such as lactoferrin (LTF), clusterin and fatty acid synthase (FASN) (Table 1a). Exosomes isolated from post-lactation purified epithelial cells also show abundant 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 was also observed using different differentiation conditions.

[0170] [Table 4]

[0171] [Table 5]

[0172] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

Claims

1. 1. A method for producing a mammalian milk-like product, comprising: A) culturing mammary epithelial cells in a culture medium to generate lactocytic mammary-like glandular organoids; B) secreting said mammalian milk-like product from said lactocytes.

2. 10. The method of claim 1, wherein the duration of step A) is 14 days or less, optionally 14 days.

3. The method of claim 1, wherein the lactocytic mammary-like glandular organoids from step A) express one or more mammary positive cell markers, optionally selected from KRT-18 and ER.

4. 2. The method of claim 1, wherein the lactocytic mammary-like gland organoids from step A) have increased mRNA expression of one or more milk bioactivity markers after induction compared to before induction.

5. The method of claim 1, wherein the lactocytic mammary gland organoids from step A) have increased LTF mRNA expression after induction compared to before induction.

6. The method of claim 1, wherein the lactocytic mammary gland organoids from step A) have increased MFGE8 mRNA expression after induction compared to before induction.

7. 10. The method of claim 1, wherein the culture medium is MammoCult medium in a suitable three-dimensional culture system.

8. Step A) further comprises: i) culturing the mammary epithelial cells; and ii) inducing expression of the milk protein.

9. 9. The method of claim 8, wherein step A i) is for no more than 7 days, optionally 7 days.

10. 9. The method of claim 8, wherein step A ii) is for no more than 7 days, optionally 7 days.

11. Step A) further comprises: i) culturing the mammary epithelial cells for 7 days in complete MammoCult medium containing basal medium, growth supplements, and supplemented with heparin and hydrocortisone; ii) inducing milk protein expression by incubating the cells in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol for 7 days.

12. Step A) further comprises: i) culturing the mammary epithelial cells for 7 days in MammoCult B medium supplemented with MammoCult growth supplement, hydrocortisone, and heparin; ii) inducing milk protein expression by incubation for 7 days in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol.

13. 10. The method of claim 1, optionally comprising step C) further processing the milk-like product to produce a modified mammalian milk-like product.

14. The method according to any one of claims 1 to 13, wherein the mammary epithelial cells are human mammary epithelial cells.

15. A human milk-like product obtainable according to the method of claim 14.

16. 16. The human milk-like product of claim 15 for use in therapy.

17. 16. The human milk-like product of claim 15 for use as a human milk substitute, optionally as a breast milk substitute.

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