Method for producing milk-like products

Abstract

The present invention provides isolated human breast milk exosomes and compositions thereof. The invention also provides an in vitro method for producing the exosome, comprising generating lactating cells derived from human induced pluripotent stem cells (hiPSCs), expressing a human milk-like product from the lactating cells, and purifying the exosome from the human milk-like product.

Description

Technical Field

[0001] This invention relates to isolated human breast milk exosomes and compositions thereof. The invention also relates to an in vitro method for producing said exosomes, the method comprising culturing and differentiating lactating cells derived from human induced pluripotent stem cells (hiPSCs), and / or mammary gland-like organoids containing such lactating cells, expressing human milk-like products from such lactating cells and / or mammary gland-like organoids, and purifying exosomes from said human milk-like products. 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, one object of the present invention is to provide an improved method for producing human milk and key components of human milk (such as exosomes) using cultured cells. Another object of the present invention is to prepare customized human milk-like products in cultured cells that can be adapted to the specific needs of the recipient and / or produce human milk bioactive substances (such as exosomes) to supplement existing cow's milk-based infant nutrition solutions. 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 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).

[0009] This article also provides a population of exosomes, wherein the population comprises exosomes as described in any part of this article.

[0010] This document also provides a composition comprising exosomes or exosome populations as described in any part of this document.

[0011] This article also provides an in vitro method for producing human breast milk exosomes, the method comprising:

[0012] A) Generate mammary gland-like organoids derived from human induced pluripotent stem cells (hiPSCs).

[0013] B) The lactating cells secrete human milk-like products, and

[0014] C) Purify the exosomes from the human milk-like product to remove impurities, optionally by chromatography, filtration, or ultracentrifugation, to separate the exosomes.

[0015] Step A) includes culturing the hiPSCs in a culture medium containing BMP4 and / or RA.

[0016] This article also provides a human breast milk exosome product that can be obtained according to the methods described in any part of this article.

[0017] Finally, this document provides a method for producing human milk fortifiers, comprising performing the methods described in any part of this document. Detailed Implementation

[0018] definition

[0019] In the context of this invention, the term "in vitro" means taking place or occurring in a test tube, petri dish, bioreactor, or elsewhere outside a living organism.

[0020] 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.

[0021] In the context of this invention, the term "lactating cell" or "mammary gland-like cell" refers to secretory epithelial cells that express the CK18 cell marker and are derived from mammalian induced pluripotent stem cells (miPSCs), particularly human induced pluripotent stem cells (hiPSCs). As used herein, human induced pluripotent stem cells (hiPSCs) are commercially available and can be selected from any suitable hiPSC cell line. In the context of this invention, a suitable human induced pluripotent stem cell line is, for example, hiPSC cell line 603, which, as used according to the invention, is commercially available from Fujifilm Cellular Dynamics (FCDI). Other suitable hiPSCs may also be selected as described by Ying Qu et al. (2017, ibid.). In one embodiment of the invention, the hiPSCs are unengineered. In one embodiment, they are unengineered as comprising exogenous nucleic acids and / or an inducible gene expression system comprising exogenous nucleic acids, wherein the inducible gene expression system is configured to express hormones or signal transduction factors. In one embodiment, the exogenous nucleic acid and / or the inducible gene expression system comprising exogenous nucleic acids promotes cell differentiation into lactating cells.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] "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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Modified human milk-like products can include standard human milk-like products and non-standard human milk-like products.

[0031] In the context of this invention, the term "EB" refers to an embryoid.

[0032] In the context of this invention, the term "mEB" refers to "embryomorph cultured in MammoCult medium".

[0033] MammoCult medium is a serum-free medium containing basal medium, at least one proliferation supplement, heparin, and hydrocortisone.

[0034] In the context of this invention, the terms “embryomorph (EB),” “mEB cultured in MammoCult medium,” “mammary globulus,” and / or “globulus” refer to a three-dimensional aggregate formed by pluripotent stem cells (PSCs) in suspension during step A) of the method of this invention.

[0035] 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.

[0036] 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.

[0037] The term "full-term baby" refers to a baby born at full term or 37 weeks of gestation or greater.

[0038] The term "premature baby" refers to a baby born at a gestational age of less than 37 weeks.

[0039] In the context of this invention, the term "birth weight" refers to the initial weight of a fetus or newborn obtained after birth.

[0040] 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).

[0041] 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).

[0042] 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).

[0043] 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.

[0044] In the context of this invention, the term "toddler" or "walking child" refers to a child aged between 1 and 3 years.

[0045] 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.

[0046] "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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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).

[0051] The emulsion fortifier according to the present invention can be in powder or liquid form.

[0052] 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.

[0053] In addition, liquid formulations are easier to mix with the composition to be fortified, while powder formulations may clump in some cases.

[0054] 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.

[0055] In the context of this invention, the term "increasing the differentiation efficiency and maturation of mammalian induced pluripotent stem cells (miPSCs) into mammary gland progenitor cells" refers to increasing the proportion of mammary gland progenitor cells relative to non-mammary gland progenitor cells derived from a miPSC initiation population that has undergone a differentiation protocol. In this invention, the differentiation protocol includes the use of BMP4 and RA. Therefore, the increase in the proportion of mammary gland progenitor cells can be compared to the proportion of mammary gland progenitor cells relative to non-mammary gland progenitor cells derived from a miPSC initiation population that has undergone a differentiation protocol excluding the use of BMP4 and RA but otherwise identical.

[0056] In the context of this invention, the term "increased viability" refers to increasing the number of living and healthy cells. These cells are capable of further differentiation steps, such as developing into mammary organoids, in the context of this invention.

[0057] In the context of this invention, the term "mammary gland progenitor cell" or similar term refers to a cell expressing at least two mammary gland progenitor cell markers. These markers include, but are not limited to, CD49f, EpCAM, MUC1, and GATA3. Conversely, in the context of this invention, the term "non-mammary gland progenitor cell" or similar term refers to a cell that does not express at least two mammary gland progenitor cell markers.

[0058] The EpiCult or EpiCultB medium mentioned in this article refers to a serum-free medium containing hydrocortisone, insulin, FGF10, and HGF.

[0059] 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.

[0060] Methods and uses

[0061] This invention relates to a method for producing mammary gland cells using iPSCs, which are cultured and differentiated under specific conditions described below, and to a method for using the mammary gland cells to produce a human milk-like product in vitro, wherein exosomes are purified from the human milk-like product in order to isolate the exosomes.

[0062] Unexpectedly, this invention has demonstrated that the addition of bone morphogenetic protein 4 (BMP4) and / or retinoic acid (RA) to methods as described in any part of this document improves the efficiency of differentiation protocols and enhances the yield and quality of terminal milk-like products. More specifically, BMP4 and / or RA have been shown to increase the differentiation efficiency of iPSCs into mammary gland progenitor cells, thereby increasing the number of mammary gland progenitor cells generated by methods as described in any part of this document. The benefits include a higher yield of mammary gland cells. BMP4 and RA have been shown to increase the expression and secretion levels of milk-specific bioactive markers such as osteopontin (OPN). It has also been shown that the increased differentiation efficiency due to the combination of BMP4 and RA can shorten the overall length of the differentiation protocol. This has significant benefits, including reduced cell death, increased milk-like product yield, and cost savings.

[0063] Mammary mammary cell production

[0064] In one aspect, the present invention relates to a method for producing mammary gland cells using iPSCs cultured and differentiated under specific conditions.

[0065] Therefore, the present invention provides a method for producing a population of mammary gland cells, the method comprising:

[0066] i) Culture mammalian induced pluripotent stem cells (miPSCs) in a medium containing bone morphogenetic protein 4 (BMP4) and / or retinoic acid (RA) to generate embryoid bodies (EBs), and

[0067] ii) Allow the EB to grow to generate a population of mammary cells.

[0068] In some embodiments, the present invention provides a method for producing a population of mammary gland cells, comprising:

[0069] i) Culture mammalian induced pluripotent stem cells (miPSCs) in a medium containing bone morphogenetic protein 4 (BMP4) to generate embryoid bodies (EBs), and

[0070] ii) Allow the EB to grow to generate a population of mammary cells.

[0071] Therefore, the present invention provides a method for producing a population of mammary gland cells, the method comprising:

[0072] i) Culture mammalian induced pluripotent stem cells (miPSCs) in a medium containing retinoic acid (RA) to generate embryoid bodies (EBs), and

[0073] ii) Allow the EB to grow to generate a population of mammary cells.

[0074] In some embodiments, the present invention provides a method for producing a population of mammary gland cells, comprising:

[0075] i) Culture mammalian induced pluripotent stem cells (miPSCs) in a medium containing bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) to generate embryoid bodies (EBs), and

[0076] ii) Allow the EB to grow to generate a population of mammary cells.

[0077] This invention also provides the use of BMP4 and / or RA in differentiation protocols to improve the differentiation efficiency of mammalian induced pluripotent stem cells (miPSCs) into mammary gland progenitor cells. In some embodiments, this invention also provides the use of BMP4 and / or RA to improve the efficiency of mammalian milk-like product production.

[0078] In some embodiments, the mammary gland cells are human mammary gland cells. In some embodiments, the mammary gland cells form lactogenic mammary gland-like organoids. These lactogenic mammary gland-like organoids are milk-producing, i.e., capable of producing milk.

[0079] In some embodiments, BMP4 is added to the culture medium at an early stage of the differentiation process as described anywhere herein. In some embodiments, BMP4 is added to the culture medium between day 0 and day 10. In some embodiments, BMP4 is added to the culture medium between day 0 and day 6. In some embodiments, BMP4 is added to the culture medium between day 0 and day 3. Day 0 is the time point at which iPSCs are first added to the culture medium, i.e., the time point at which iPSCs are first induced to differentiate.

[0080] In some implementations, BMP4 is added to the culture medium for 3 days.

[0081] In some embodiments, BMP4 is added to the culture medium at a concentration of 5 ng / mL to 20 ng / mL. In some embodiments, BMP4 is added to the culture medium at a concentration of 5 ng / mL. In some embodiments, BMP4 is added to the culture medium at a concentration of 10 ng / mL. In some embodiments, BMP4 is added to the culture medium at a concentration of 20 ng / mL.

[0082] In some implementations, BMP4 is added to the culture medium at a concentration of 5 ng / mL to 20 ng / mL between day 0 and day 3.

[0083] In some embodiments, RA is added to the culture conditions at an early stage of the differentiation process as described anywhere herein. In some embodiments, RA is added to the culture medium during the mammary lineage orientation phase. In some embodiments, RA is added to the culture medium between day 10 and day 15. In some embodiments, RA is added to the culture medium between day 6 and day 11. In some embodiments, RA is not added to the culture medium after day 11. In some embodiments, RA is added to the culture medium before day 11. Day 0 is the time point at which iPSCs are first added to the culture medium, i.e., the time point at which iPSCs are first induced to differentiate.

[0084] In some implementations, RA is added to the culture medium for 5 days.

[0085] In some implementations, RA is added to the culture medium at a concentration of 1 µM.

[0086] In some implementations, RA is added at a concentration between 1 µM under culture conditions between day 10 and day 15.

[0087] In some implementations, BMP4 is added to the culture medium between day 0 and day 3, and RA is added to the culture medium between day 10 and day 15.

[0088] In some implementations, BMP4 is added to the culture medium between day 0 and day 3, and RA is added to the culture medium between day 6 and day 11.

[0089] EB generated in any of the methods described herein expresses one or more positive progenitor cell markers of mammary glands. In some embodiments, the one or more positive progenitor cell markers of mammary glands are selected from EpCAM, CD49f, MUC1, and GATA3.

[0090] In some implementations, the EB has increased expression of one or more breast glandular positive progenitor cell markers compared to the expression levels of the breast glandular positive progenitor cell markers in EBs not treated with BMP4.

[0091] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EB express one or more breast glandular positive progenitor cell markers. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EB express two or more breast glandular positive progenitor cell markers. In some embodiments, the breast glandular positive progenitor cell markers are selected from EpCAM, CD49f, MUC1, and GATA3.

[0092] In some embodiments, at least 35% of EBs express EpCAM and CD49f, the positive progenitor cell markers for mammary gland tissue. In some embodiments, at least 60% of EBs express EpCAM and CD49f during the intermediate differentiation stage. In some embodiments, at least 35% of EBs express EpCAM and CD49f during the pre-induction stage. In some embodiments, at least 15% of EBs express EpCAM and CD49f during the post-induction stage. In some embodiments, at least 40% of EBs express EpCAM and CD49f during the post-induction stage.

[0093] In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers. In some embodiments, at least 20% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers during the intermediate differentiation stage. In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers before induction. In some embodiments, at least 5% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers after induction. In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers after induction.

[0094] In some embodiments, at least 20% of EBs express GATA3 and EpCAM, positive progenitor cell markers for mammary gland tissue. In some embodiments, at least 50% of EBs express GATA3 and EpCAM during the intermediate differentiation stage. In some embodiments, at least 25% of EBs express GATA3 and EpCAM before induction. In some embodiments, at least 15% of EBs express GATA3 and EpCAM after induction. In some embodiments, at least 40% of EBs express GATA3 and EpCAM after induction.

[0095] In some implementations, the intermediate differentiation phase is day 25, the pre-induction phase is day 35, and the post-induction phase is day 42. In some implementations, the intermediate differentiation phase occurs around day 20. The pre-induction phase is day 26, and the post-induction phase is day 31.

[0096] In some embodiments, the EB expresses one or more milk-specific bioactive markers. In some embodiments, the milk-specific bioactive marker is osteopontin (OPN).

[0097] In some embodiments, the expression of one or more milk-specific bioactive markers in the EB is increased compared to the expression levels of the markers in EB not treated with BMP4 and / or RA. In some embodiments, the secretion of one or more milk-specific bioactive markers in the EB is increased compared to the expression levels of the markers in EB not treated with BMP4 and / or RA. In some embodiments, the OPN secretion of the EB is increased compared to the OPN expression levels in EB not treated with BMP4 and / or RA. In some embodiments, the OPN secretion of the EB is increased by 40% or more compared to the OPN expression levels in EB not treated with BMP4 and / or RA.

[0098] The methods for generating mammary gland cell populations as described anywhere herein may be combined with and / or utilized in methods for generating mammalian milk-like products as described anywhere herein, particularly as part of step A) as described anywhere herein.

[0099] For example, in some embodiments of methods for generating mammary gland cell populations as described anywhere herein, the culture step i) includes culturing miPSCs in MammoCult medium and BMP4 in a 3D suspension culture system, such as 3D suspension conditions, thereby guiding the iPSCs to differentiate into non-neuroectodermal cells. In some embodiments, step i) is performed for at least 12 days. In some embodiments, step i) is performed for no more than 8 days.

[0100] In some embodiments of the method for producing mammary gland cell populations as described in any part of this document, growth step ii) includes growing EB in a 3D embedding system containing RA (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or type I collagen). In some embodiments, step A ii) is performed for at least 30 days, e.g., 32 days. In some embodiments, step A ii) lasts for at least 23 days, e.g., no more than 25 days.

[0101] mammalian milk production

[0102] In another aspect, the present 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 optional step C) as defined herein. The method for producing mammalian milk-like products as defined herein may further comprise a method for producing mammalian mammary gland cells as part of step A). ​​Specifically, BMP4 and / or RA may be added to any of the methods for producing mammalian milk-like products as defined herein, particularly as part of step A). ​​The method also includes different time lengths for producing mammalian milk-like products.

[0103] Step A—Generation of lactating cells and / or mammary gland-like organoids from hiPSCs

[0104] According to the method of the present invention, mammary gland-like cells and / or organoid structures are generated in step A).

[0105] Such mammary gland-like cells and / or organoid structures can be generated using any published method that utilizes iPSCs.

[0106] In one implementation, such mammary gland-like cells and / or organoid structures can be generated according to the process described by Ying Qu et al. in StemCell Reports, Vol. 8, pp. 205-215 (the full text of which is incorporated herein by reference).

[0107] More precisely, the methodology described in the aforementioned scientific publications (hereinafter also referred to as “Ying Qu publications” or Ying Qu et al. (2017)) represents a two-step protocol for generating human mammary gland-like cells and / or organoids from iPSCs.

[0108] The protocol preferably includes a first step (step 1): differentiating and enriching spheroids (mEB / mammary globules) containing non-neuroectodermal cells from iPSCs, and a second step (step 2): generating mammary-like organoids from mEB (mammary globules) formed by culturing for 10 days using a mixed 3D floating gel culture of Matrigel and type I collagen.

[0109] More specifically, step A preferably includes:

[0110] -Embryoid formation

[0111] - Breast spectrum orientation

[0112] - Branching and vesicle differentiation, and

[0113] - Production of milk bioactive substances.

[0114] Each of these stages can be performed for a specific amount of time using a specific culture medium.

[0115] In some implementations, step A is performed for a total of 40 to 45 days, preferably 42 days. In some implementations, step A is performed for a total of 40 to 42 days. In some implementations, step A is performed for 41 days.

[0116] In some embodiments, step A is shortened and performed for less than 42 days, optionally less than 40 days, optionally less than 31 days. Preferably, step A is performed for 31 days. In some embodiments, step A is performed for 30 to 39 days. In some embodiments, step A is performed for 35 to 39 days. In some embodiments, step A is performed for 30 to 35 days.

[0117] In some implementations, the embryoid formation phase occurs between day 0 and day 10. In other implementations, the embryoid formation phase lasts for 10 days.

[0118] In some embodiments, the germline formation phase is shortened and occurs between day 0 and day 6. In some embodiments, the germline formation phase lasts for 6 days. In some embodiments, the germline formation phase lasts for 10 days or less.

[0119] In some embodiments, the breast lineage orientation phase occurs between day 10 and day 15. In some embodiments, the breast lineage orientation phase occurs between day 6 and day 11. In some embodiments, the germline formation phase lasts for 5 days. In some embodiments, the germline formation phase lasts for no more than 5 days.

[0120] In some implementations, the branching and vesicle differentiation phase occurs between day 15 and day 35. In other implementations, the branching and vesicle differentiation phase lasts for 20 days.

[0121] In some implementations, the branching and vesicle differentiation phase is shortened and occurs between day 11 and day 26. In some implementations, the branching and vesicle differentiation phase lasts for 15 days. In some implementations, the branching and vesicle differentiation phase does not exceed 15 days.

[0122] In some embodiments, the induction phase of milk bioactive substances occurs between day 35 and day 42. In some embodiments, the induction phase of milk bioactive substances lasts for 7 days.

[0123] In some embodiments, the induction phase of the milk bioactive substances is shortened and occurs between day 26 and day 31. In some embodiments, the induction phase of the milk bioactive substances lasts for 5 days. In some embodiments, the induction phase of the milk bioactive substances does not exceed 5 days.

[0124] Therefore, in some implementations, step A is carried out for a total of 42 days, wherein the embryoid formation phase is between day 0 and day 10, the mammary lineage orientation phase is between day 10 and day 15, the branching and vesicle differentiation phase is between day 15 and day 35, and the induction phase of milk bioactive substances is between day 35 and day 42.

[0125] In other embodiments, the process is shortened so that step A takes a total of 31 days, with the embryoid formation phase between day 0 and day 6, the mammary lineage orientation phase between day 6 and day 11, the branching and vesicle differentiation phase between day 11 and day 26, and the induction phase of milk bioactive substances between day 26 and day 31.

[0126] Further details regarding the time periods and culture media used for the methods described anywhere in this document are provided below.

[0127] In step 1, hiPSCs are cultured in complete MammoCult medium (StemCell Technologies) to differentiate and enrich spheroids (mEB / mammary globules) containing non-neuroectodermal cells from hiPSCs. Preferably, the complete MammoCult medium consists of basal medium, proliferation supplement, heparin (typically 4 µg / mL), and hydrocortisone (typically 0.48 µg / mL). The medium is typically changed every three days. The mEB (mammary globules) obtained in the above steps are then enriched with non-neuroectodermal cells.

[0128] In some implementations, BMP4 is added to the culture medium in step 1.

[0129] In some implementations, as described in any part of this document, BMP4 is added to the culture medium.

[0130] In some embodiments, BMP4 is added to the culture conditions at an early stage of the differentiation process as described in any part of this document. In some embodiments, BMP4 is added to the culture medium between day 0 and day 10. In some embodiments, BMP4 is added to the culture medium between day 0 and day 6. In some embodiments, BMP4 is added to the culture medium between day 0 and day 3. Day 0 is the time point at which iPSCs are first added to the culture medium, i.e., the time point at which iPSCs are first induced to differentiate.

[0131] In some implementations, BMP4 is added to the culture medium for 3 days.

[0132] In some embodiments, BMP4 is added to the culture medium at a concentration of 5 ng / mL to 20 ng / mL. In some embodiments, BMP4 is added to the culture medium at a concentration of 5 ng / mL. In some embodiments, BMP4 is added to the culture medium at a concentration of 10 ng / mL. In some embodiments, BMP4 is added to the culture medium at a concentration of 20 ng / mL.

[0133] In some implementations, BMP4 is added to the culture medium at a concentration of 5 ng / mL to 20 ng / mL between day 0 and day 3.

[0134] In step 2, following the protocol of Ying Qu et al. (2017), mammary organoids were generated by first preparing 3D cultures based on a mixed floating gel (e.g., Matrigel and type I collagen). Then, mEBs (mammary globules) formed in 10 days were grown for 5 days in a mixed gel floating in complete EpiCultB medium supplemented with parathyroid hormone (pTHrP). Cells were then cultured in complete EpiCultB medium supplemented with hydrocortisone, insulin, FGF10, and HGF to induce branching and vesicle differentiation for mammary organoid / lactogenic cells. Milk protein expression was typically induced on day 35 by adding prolactin, hydrocortisone, and insulin to complete EpiCultB medium supplemented with BSA (lactogenic medium) and culturing for 5 days. The method of Ying Qu et al. (2017) typically achieved expression on day 40.

[0135] In some implementations, RA is added to the culture medium in step 2.

[0136] In some implementations, as described in any part of this document, RA is added to the culture medium.

[0137] In some embodiments, RA is added to the culture medium at an early stage of the differentiation process as described in any part of this document. In some embodiments, RA is added to the culture medium during the mammary lineage orientation phase. In some embodiments, RA is added to the culture medium between day 10 and day 15. In some embodiments, RA is added to the culture medium between day 6 and day 11. In some embodiments, RA is not added to the culture medium after day 11. Day 0 is the time point at which iPSCs are first added to the culture medium, i.e., the time point at which iPSCs are first induced to differentiate.

[0138] In some implementations, RA is added to the culture medium for 5 days.

[0139] In some implementations, RA is added to the culture medium at a concentration of 1 µM.

[0140] In some implementations, RA is added to the culture medium at a concentration between 1 µM and 15 days between day 10 and day 15.

[0141] In one embodiment of the invention, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0142] i) iPSCs were cultured in a suitable medium (e.g., MammoCult medium) and BMP4 as described anywhere herein, guiding the differentiation of iPSCs into non-neural ectodermal cells, and collecting the resulting mammary globules after 10 days; and

[0143] ii) Allow such mammary globules to grow for at least 10 days to generate lactating cells in a suitable system containing RA as described anywhere herein (e.g., a hybrid floating gel culture system as described in Hassiotou F. et al. StemCells, 2012).

[0144] In one embodiment of the invention, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0145] i) iPSCs were cultured in a suitable medium (e.g., MammoCult medium) and BMP4 as described in any part of this document, guiding their differentiation into non-neuroectodermal cells, and the resulting mammary globules were collected after 6 days; and

[0146] ii) In a suitable system containing RA as described anywhere herein (e.g., a hybrid floating gel culture system as described by Hassiotou F. et al. StemCells, 2012), such mammary globules are grown for less than 10 days to generate lactating cells.

[0147] In another embodiment, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0148] i) Inducing iPSC differentiation into non-neuroectodermal cells by culturing iPSCs in a suitable medium (e.g., MammoCult medium) and BMP4 under non-adherent mammary bulb formation conditions as described anywhere herein; and

[0149] ii) Allow such mammary globules to grow for at least 10 days in a suitable 3D system containing RA as described anywhere herein (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen, or a suspension culture in a non-adherent culture dish) to generate lactating cells.

[0150] In another embodiment, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0151] i) Inducing iPSC differentiation into non-neuroectodermal cells by culturing iPSCs in a suitable medium (e.g., MammoCult medium) and BMP4 under non-adherent mammary bulb formation conditions as described anywhere herein; and

[0152] ii) To generate lactating cells, such mammary globules are grown for less than 10 days in a suitable 3D system containing RA as described anywhere herein, such as a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen, or a suspension culture in a non-adherent culture dish.

[0153] In one embodiment, mammary gland orientation in step A) is achieved by applying a culture medium (e.g., EpiCultB) conditioned with specific factors (e.g., parathyroid hormone (pTHrP), hydrocortisone, insulin, FGF10, and HGF) and RA.

[0154] In one embodiment of the present invention, the method includes generating a mammary gland-like organoid in step A).

[0155] In one embodiment of the invention, the method for generating mammary-like organoids in step A) includes culturing the cells under conditions selected from the group consisting of: 2D monolayer cells, 2D EBs with adhesion, suspended in non-adherent culture plates and in mixed floating gels.

[0156] In a preferred embodiment, the mixed floating gel comprises Matrigel and type I collagen.

[0157] In another preferred embodiment, the mammary globules (mEB) in step A) are cultured for at least 15 days in a suitable system (e.g., a hybrid floating gel culture system described in Hassiotou F. et al. Stem Cells, 2012).

[0158] In a more preferred embodiment, the mammary globules (mEB) in step A) are cultured for at least 20 days in a suitable system (e.g., a hybrid floating gel culture system described in Hassiotou F. et al. Stem Cells, 2012).

[0159] In one embodiment, the method according to the invention provides culture conditions according to step A) [e.g., in step A)i) and / or step A)ii)], which are suitable for generating lactating cells derived from human induced pluripotent stem cells (hiPSCs) capable of secreting human milk-like products.

[0160] In a preferred embodiment, a method for producing human milk-like products is provided, comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein step A) includes guiding the differentiation of hiPSCs into mammary gland cells (e.g., lactating cells) in a suitable 3D culture system (e.g., 3D suspension conditions) containing BMP4 and / or RA as described in any part of this document. In some embodiments, this process lasts for at least 42 days. In some embodiments, it lasts for no more than 31 days. In some embodiments, it lasts for at least 31 days.

[0161] In another preferred embodiment, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0162] i) By culturing hiPSCs in a suitable 3D culture system (e.g., under 3D suspension conditions) in a suitable medium (e.g., MammoCult medium) and BMP4 as described anywhere herein for at least 12 days (days -2 to 10), hiPSCs are guided to differentiate into non-neural ectodermal cells, and

[0163] ii) The formed mEBs (mammary globules) are grown for at least 30 days, preferably 32 days, in a suitable 3D embedding system containing RA as described elsewhere herein (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or type I collagen) to generate lactating cells.

[0164] In another preferred embodiment, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0165] i) By culturing hiPSCs in a suitable 3D culture system (e.g., 3D suspension conditions) in a suitable medium (e.g., MammoCult medium) and BMP4 as described elsewhere herein for no more than 8 days (days -2 to 6), hiPSCs are guided to differentiate into non-neural ectodermal cells.

[0166] ii) In a suitable 3D embedding system containing RA as described elsewhere herein, for example, in a mixed floating gel composed of matrix proteins such as Matrigel and / or type I collagen, the formed mEB (mammary globules) are grown for no more than 25 days to generate lactating cells.

[0167] In a particularly preferred embodiment of the present invention, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein step A)i) is defined as follows:

[0168] i) via standard iPSC medium E8 (containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenide, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, as described in Chen et al., Nat Methods, 2011) or mTeSR ™ Embryoids (EBs) were generated from hiPSCs through two-day incubation (day -2 to day 0), and highly enriched mEBs (mammaglobules) of non-neuroectoderm cells were produced by incubating the EBs for 10 days (day 0 to day 10) in complete MammoCult medium (StemCell Technologies) containing basal medium, proliferation supplements, and supplemented with heparin (typically 4 µg / mL), hydrocortisone (typically 0.48 µg / mL), and BMP4 as described in any part of this document.

[0169] Step A)ii) is further divided into sub-steps and includes the following steps ii), iii) and iv):

[0170] ii) Incubate mEB (mammary globules) in complete EpiCult B medium supplemented with EpiCult proliferation supplement and parathyroid hormone (pTHrP) for 5 days (days 10 to 15).

[0171] iii) Incubation of mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 20 consecutive days (days 15 to 35) promoted branching and vesicle differentiation and mammary cell specialization.

[0172] iv) Milk protein expression was induced by incubating mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days (days 35 to 42).

[0173] In a particularly preferred embodiment of the present invention, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein step A)i) is defined as follows:

[0174] i) via standard iPSC medium E8 (containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenide, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, as described in Chen et al., Nat Methods, 2011) or mTeSR ™ Embryoids (EBs) were generated from hiPSCs through two-day incubation (day -2 to day 0), and highly enriched mEBs (mammaglobules) of non-neuroectoderm cells were produced by incubating the EBs for 6 days (day 0 to day 6) in complete MammoCult medium (StemCell Technologies) containing basal medium, proliferation supplements, and supplemented with heparin (typically 4 µg / mL), hydrocortisone (typically 0.48 µg / mL), and BMP4 as described in any part of this document.

[0175] Step A)ii) is further divided into sub-steps and includes the following steps ii), iii) and iv):

[0176] ii) Incubate mEB (mammary globules) in complete EpiCult B medium supplemented with EpiCult proliferation supplement and parathyroid hormone (pTHrP) and RA for 5 days (days 6 to 11).

[0177] iii) Incubation of mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 15 days (days 11 to 26) promoted branching and vesicle differentiation as well as mammary cell specialization.

[0178] iv) Milk protein expression was induced by incubating mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 5 days (days 26 to 31).

[0179] Step iv) preferably leads to the differentiation of milk protein-expressing cells, especially lactating cells and / or mammary gland-like organoids.

[0180] In another particularly preferred embodiment of the invention, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein step A)i) is defined as follows:

[0181] i) via standard iPSC medium E8 (containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenide, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, as described in Chen et al., Nat Methods, 2011) mTeSR ™ Embryoids (EBs) were generated from hiPSCs by incubation for two days (day-2 to day 0), and highly enriched mEBs (mammaglobules) of non-neuroectoderm cells were generated by incubating the EBs in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin and BMP4 as described in any part of this document, wherein step A)ii) is further divided into sub-steps and includes the following steps ii), iii) and iv):

[0182] ii) The formed mEB (mammary globules) were embedded in a mixture of Matrigel and type I collagen floating in EpiCultB medium supplemented with EpiCult proliferative supplement and parathyroid hormone (pTHrP) and RA for 5 days (days 10 to 15).

[0183] iii) Incubation of embedded mEBs (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 20 days (days 15 to 35) promoted branching and vesicle differentiation as well as mammary cell specialization.

[0184] iv) Milk protein expression was induced by incubating mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days (days 35 to 42).

[0185] In another particularly preferred embodiment of the invention, a method for producing human milk-like products is provided, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein step A)i) is defined as follows:

[0186] i) via standard iPSC medium E8 (containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenide, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, as described in Chen et al., Nat Methods, 2011) mTeSR ™ Embryoids (EBs) were generated from hiPSCs by incubation for two days (day-2 to day 0), and highly enriched mEBs (mammaglobules) of non-neuroectoderm cells were generated by incubating the EBs in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin and BMP4 as described in any part of this document, wherein step A)ii) is further divided into sub-steps and includes the following steps ii), iii) and iv):

[0187] ii) The formed mEB (mammary globules) were embedded in a mixture of Matrigel and type I collagen floating in EpiCultB medium supplemented with EpiCult proliferative supplement and parathyroid hormone (pTHrP) and RA for 5 days (days 6 to 11).

[0188] iii) Incubation of embedded mEBs (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 15 days (days 11 to 26) promoted branching and vesicle differentiation and mammary cell specialization.

[0189] iv) Milk protein expression was induced by incubating mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 5 days (days 26 to 31).

[0190] Step iv) preferably leads to the differentiation of milk protein-expressing cells, especially lactating cells and / or mammary gland-like organoids.

[0191] The standard iPSC medium E8 mentioned in this article (containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenide, FGF2, insulin, NaHCO3, and transferrin, TGFβ1, or NODAL, as described by Chen et al., Nat Methods, 2011) is commercially available, for example as “Essential 8”. ™ The culture medium was purchased from ThermoFischer Scientific, catalog number A1517001 (see also...) https: / / www.thermofisher.com / order / catalog / product / A1517001# / A1517001 ).

[0192] mTeSR ™ Culture media are commercially available from STEMCELL Technologies, catalog number 85850 (see also...) https: / / www.stemcell.com / mtesr1.html This type of culture medium is also described in "Defined, Feeder-Independent medium for human hembryonic stem cell culture" in Current Protocols in Stem Cell Biology, Volume 2, Issue 1, September 2007.

[0193] In one embodiment, steps iii) and / or iv) as defined above for a particularly preferred embodiment preferably result 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.

[0194] In another embodiment, after inducing mEB (mammary bulbs) in steps ii) and / or iv) as defined above for a particularly preferred embodiment, mammary gland organoids expressing one or more of the following markers: β-casein, milk proteins, and hormone receptors; luminal cells expressing one or more of the following markers: EpCAM, MUC1, CD49F, GATA3, CK8, and CK18; and basal cells expressing one or more of the following markers: CK14, α-smooth muscle actin, and P63.

[0195] In one embodiment of the present invention, the above-described method for producing human milk-like products is provided.

[0196] 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.

[0197] When BMP4 and / or RA are added to a culture medium in a method for producing mammalian milk-like products as described in any part of this document, the EB generated in the method as described in any part of this document expresses one or more positive progenitor cell markers of mammary glands. In some embodiments, the one or more positive progenitor cell markers of mammary glands are selected from EpCAM, CD49f, MUC1, and GATA3.

[0198] In some implementations, the EB has increased expression of one or more breast glandular positive progenitor cell markers compared to the expression levels of the breast glandular positive progenitor cell markers in EBs not treated with BMP4.

[0199] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EB express one or more breast glandular positive progenitor cell markers. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EB express two or more breast glandular positive progenitor cell markers. In some embodiments, the breast glandular positive progenitor cell markers are selected from EpCAM, CD49f, MUC1, and GATA3.

[0200] In some embodiments, at least 35% of EBs express EpCAM and CD49f, the positive progenitor cell markers for mammary gland tissue. In some embodiments, at least 60% of EBs express EpCAM and CD49f during the intermediate differentiation stage. In some embodiments, at least 35% of EBs express EpCAM and CD49f during the pre-induction stage. In some embodiments, at least 40% of EBs express EpCAM and CD49f during the post-induction stage.

[0201] In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers. In some embodiments, at least 20% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers during the intermediate differentiation stage. In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers before induction. In some embodiments, at least 5% of EBs express MUC1 and EpCAM mammary gland-positive progenitor cell markers after induction.

[0202] In some embodiments, at least 20% of EBs express GATA3 and EpCAM, positive progenitor cell markers for mammary gland tissue. In some embodiments, at least 50% of EBs express GATA3 and EpCAM during the intermediate differentiation stage. In some embodiments, at least 25% of EBs express GATA3 and EpCAM during the pre-induction stage. In some embodiments, at least 15% of EBs express GATA3 and EpCAM during the post-induction stage.

[0203] In some implementations, the intermediate differentiation phase occurs on day 25. The pre-induction phase occurs on day 35, and the post-induction phase occurs on day 42. In some implementations, the intermediate differentiation phase occurs between days 20. The pre-induction phase occurs on day 26, and the post-induction phase occurs on day 31.

[0204] In some embodiments, the EB expresses one or more milk-specific bioactive markers. In some embodiments, the milk-specific bioactive marker is osteopontin (OPN).

[0205] In some implementations, the expression of one or more milk-specific bioactive markers in the EB is increased compared to the expression levels of the non-neuronal ectodermal markers in the EB that were not treated with BMP4 and / or RA.

[0206] Therefore, for example, this article provides a method for producing human milk-like products, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0207] i) iPSCs were cultured in a suitable medium (e.g., MammoCult medium) and BMP4 as described anywhere herein, guiding the differentiation of iPSCs into non-neural ectodermal cells, and collecting the resulting mammary globules after 10 days; and

[0208] ii) Allow such mammary globules to grow for at least 10 days in a suitable system (e.g., a hybrid floating gel culture system as described by Hassiotou F. et al., Stem Cells, 2012) to generate lactating cells.

[0209] Compared to the expression levels of the positive progenitor cell markers of mammary glands in untreated mammary globules (EBs), the EBs exhibit increased expression of one or more positive progenitor cell markers of mammary glands.

[0210] Therefore, for example, this article provides a method for producing human milk-like products, the method comprising: generating lactating cells from human induced pluripotent stem cells (hiPSCs) in step A), wherein such step A) includes:

[0211] i) iPSCs were cultured in a suitable medium (e.g., MammoCult medium) and BMP4 as described in any part of this document, guiding their differentiation into non-neuroectodermal cells, and the resulting mammary globules were collected after 6 days; and

[0212] ii) To generate lactating cells, such mammary globules are grown for less than 10 days in a suitable system containing RA (e.g., a hybrid floating gel culture system described in Hassiotou F. et al. Stem Cells, 2012).

[0213] The expression of one or more positive progenitor cell markers of the mammary glands in the blastoglobules (EB) was increased compared with the expression levels of the positive progenitor cell markers of the mammary glands in the blastoglobules (EB) that were not treated with BMP4 and / or RA.

[0214] 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.

[0215] Step B—Expression of human breast milk-like product

[0216] In one embodiment of the invention, the method includes expressing human milk-like products from mammary gland-like organoids derived preferably from human induced pluripotent stem cells (hiPSCs) prepared according to step A). ​​Preferably, the expression of human milk-like products occurs after inducing expression of human milk-like products from such lactating cells and / or mammary gland-like organoids.

[0217] 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).

[0218] Specifically, the human milk-like product obtained from mammary gland-like organoids derived from human induced pluripotent stem cells (hiPSCs) preferably prepared according to step A) contains bioactive substances found in human milk, selected from or consisting of the group consisting of proteins, lipids, oligosaccharides, preferably oligosaccharides, etc. Specifically, the inventors have sought to maintain consistency with particularly preferred formulations according to steps A) i) to iv) performed as described above, particularly regarding 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).

[0219] 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) 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.

[0220] In another embodiment, the human milk-like product obtained from a mammary gland-like organoid derived preferably from human induced pluripotent stem cells (hiPSCs) prepared according to step A) contains bioactive substances from human milk, selected from the group consisting of or comprising: 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-16:1 n7 fatty acids, C-17:0 fatty acids, C-18:0 fatty acids, C-18:1 n9 fatty acids, C-18:1 fatty acids, C-18:2 n6 fatty acids, C-20:0 fatty acids, C-20:1 n9 fatty acids, C-18:3 n3 fatty acids, C-22:0 fatty acids, lactoferrin, albumin, prolactin, α-lactalbumin, and other bioactive substances. 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.

[0221] In one embodiment of the invention, the human milk-like product obtained from mammary gland-like organoids derived from human induced pluripotent stem cells (hiPSCs) is a standard human milk product. In another embodiment of the invention, the human milk-like product obtained from mammary gland-like organoids derived from human induced pluripotent stem cells (hiPSCs) is a non-standard human milk product.

[0222] Step C—Further processing to produce a modified human breast milk-like product.

[0223] 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.

[0224] 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).

[0225] In particular, in some embodiments, the human 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 isolated human milk-like products.

[0226] In some embodiments, the isolated human milk-like product is formulated as a composition. In some embodiments, the isolated human milk-like product is formulated as a nutritional composition.

[0227] In some embodiments, specific bioactive substances from human milk-like products are extracted, isolated, and purified, selected from or consisting of the group consisting of oligosaccharides, lipids, proteins, exosomes, and miRNAs. In particular, exosomes are extracted, isolated, and purified from human milk-like products. Therefore, the present invention provides isolated bioactive substances from human milk-like products, selected from or consisting of the group consisting of oligosaccharides, lipids, proteins, exosomes, and miRNAs. In particular, the present invention provides isolated exosomes.

[0228] In some embodiments, the isolated bioactive substances from human milk-like products are formulated into compositions. In some embodiments, the isolated bioactive substances from human milk-like products are formulated into nutritional compositions.

[0229] In some embodiments, the isolated human milk-like exosomes are formulated as a composition. In some embodiments, the isolated human milk-like exosomes are formulated as a nutritional composition.

[0230] 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.

[0231] In some implementations, the chromatography method is column-based chromatography, such as size inclusion or size exclusion chromatography.

[0232] 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.

[0233] In some implementations, step C) further includes sterilizing the isolated exosomes.

[0234] In some implementations, step C) further includes storing the isolated exosomes at a temperature below freezing (optionally -80°C).

[0235] 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.

[0236] In some implementations, the method also includes formulating isolated exosomes together with supplemental nutritional ingredients.

[0237] In some implementations, the method also includes aliquoting the isolated exosomes into containers for consumption.

[0238] Human milk-like products

[0239] "Standard" human breast milk-like products

[0240] 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.

[0241] 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.

[0242] In such implementations, human breast milk-like products can be used as a substitute for breastfeeding when actual breastfeeding is not possible.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] In one embodiment, the human milk-like product according to the invention is not a product of naturally occurring human breast milk secretion.

[0247] In one implementation, human breast milk-like products are used to provide optimal nutrition for infants.

[0248] In one implementation, human breast milk-like products are used to promote the healthy growth of infants.

[0249] In one implementation, human breast milk-like products are used to prevent infant infections and obesity, as well as to promote infant immune development.

[0250] In one implementation, the human breast milk-like product is an unmodified human breast milk-like product.

[0251] In another embodiment, the human breast milk-like product is a modified human breast milk-like product.

[0252] In one embodiment, the human milk-like product according to the invention comprises: proteins, lipids, carbohydrates, vitamins, and minerals.

[0253] In another embodiment, the human milk-like product according to the present invention comprises: proteins, lipids, carbohydrates, vitamins, minerals, and bioactive substances.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] In one implementation, the human breast milk-like product contains probiotics.

[0258] 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.

[0259] In such implementations, human breast milk-like products can be used to optimize gastrointestinal function and / or promote immunity.

[0260] In one implementation, the human breast milk-like product contains secretory IgA and probiotics.

[0261] 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.

[0262] Non-standard human breast milk-like products

[0263] 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".

[0264] 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.

[0265] Human milk fortifiers and human milk bioactive supplements

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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).

[0271] 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).

[0272] 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.

[0273] 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).

[0274] In such implementations, human breast milk-like products can be used to optimize gastrointestinal function and / or promote immunity.

[0275] Human breast milk-like products for infants with genetic diseases

[0276] 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.

[0277] Galactosemia

[0278] 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.

[0279] 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.

[0280] 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).

[0281] In another embodiment, according to the method of the present invention, hiPSCs can be generated using GMO α-lactalbumin-deficient human cells in step A) to obtain a human breast milk-like product that is lactose-free and / or contains lactose sugars.

[0282] phenylketonuria

[0283] 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.

[0284] In such implementations, human breast milk-like products should contain no or have reduced levels of phenylalanine.

[0285] In such implementations, human breast milk-like products can be used to promote the healthy growth of infants affected by PKU.

[0286] 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.

[0287] 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).

[0288] 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).

[0289] 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.

[0290] Components of human milk-like products

[0291] 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.

[0292] exosomes

[0293] 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.

[0294] 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.

[0295] 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.

[0296] The exosomes of this invention are not produced from naturally occurring human breast milk.

[0297] 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.

[0298] Therefore, for example, the present invention relates to an in vitro method for producing exosomes, the method comprising:

[0299] A) Generate mammary gland-like organoids derived from human induced pluripotent stem cells (hiPSCs).

[0300] B) The lactating cells secrete human milk-like products, and

[0301] C) Purify the exosomes from the human milk sample by chromatography, filtration, or ultracentrifugation to isolate the exosomes.

[0302] Step A) involves culturing the hiPSC in a medium containing BMP4 and / or RA.

[0303] Optionally, step A) is performed for a total of 42 or 31 days, and / or

[0304] Step A) is performed under 3D suspension conditions.

[0305] The exosomes of this invention differ in composition from those derived from natural human breast milk.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] Exosomal proteomics

[0312] Through proteomics analysis, approximately 2,000 proteins have been identified in human breast milk exosomes in the literature.

[0313] 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.

[0314] In particular, it was found that the exosomes of the present invention lack one or more proteins present in exosomes derived from natural human breast milk. 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).

[0315] 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.

[0316] 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).

[0317] In some implementations, the exosome does not contain C4a anaphylatoxin (CFA).

[0318] In some implementations, the exosome does not contain hypoxia upregulated protein 1 (HYOU1).

[0319] In some implementations, the exosome does not contain apolipoprotein A-IV (APOA4).

[0320] In some implementations, the exosome does not contain procollagen-lysine, 2-ketoglutarate 5-dioxygenase 1 (PLOD1).

[0321] In some implementations, the exosome does not contain cytoplasmic threonine-tRNA ligase 1 (TARS1).

[0322] In some implementations, the exosome does not contain a cytoplasmic nonspecific dipeptidase (CNDP2).

[0323] 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).

[0324] 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.

[0325] Table 1

[0326]

[0327] 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.

[0328] 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.

[0329] 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.

[0330] As described herein, in some embodiments, the present invention relates to a composition comprising exosomes or exosome populations as described in any part herein. In other embodiments, the present invention relates to a 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: 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).

[0331] Exosomal miRNA profile

[0332] 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.

[0333] 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.

[0334] 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.

[0335] In some implementations, the exosome comprises miR-148a-3p, miR-22-3p, miR-125b-5b, and miR-6126.

[0336] 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.

[0337] 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.

[0338] Exosomal lipidomics

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] In some embodiments, the exosome contains cholesterol esters, ceramides, triglycerides, diglycerides, total phospholipids, alkyl phospholipids, and alkenyl phospholipids.

[0344] In some embodiments, the exosome comprises one or more phospholipids selected from sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0345] In some embodiments, the exosome contains sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0346] 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.

[0347] 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.

[0348] Exosome size

[0349] 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.

[0350] 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.

[0351] In some implementations, the diameter of the exosome is 65 nm to 75 nm.

[0352] 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.

[0353] Additional embodiments of the present invention

[0354] This invention can be described according to the following numbered statements:

[0355] S1. Isolated human breast milk exosomes, wherein the exosomes do 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).

[0356] S2. The exosomes according to statement 1, wherein the exosomes comprise lactoferrin (LTF), annexin A2 (ANXA2), lactoglucin (MFGE8), MUC1, and tetraspan proteins CD9, CD81, and CD63.

[0357] S3. The exosomes according to statement 1 or 2, wherein the exosomes comprise one or more miRNAs selected from miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

[0358] S4. The exosomes according to any of the foregoing statements, wherein the exosomes comprise one or more lipids selected from cholesterol esters, ceramides, triglycerides, diglycerides, lysophospholipids, total phospholipids, alkyl phospholipids and alkenyl phospholipids.

[0359] S5. The exosome according to any of the foregoing statements, wherein the exosome comprises one or more phospholipids selected from sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine.

[0360] S6. The exosomes according to any of the foregoing statements, wherein the diameter of the exosomes is 50 nm to 100 nm.

[0361] S7. The exosomes according to statement 6, wherein the diameter of the exosomes is 65 nm to 75 nm.

[0362] S8. The exosomes according to any of the foregoing statements, wherein the exosomes have the same function as exosomes derived from natural human breast milk.

[0363] S9. The exosome according to any of the foregoing statements, wherein the exosome has the same structure as the exosome derived from natural human breast milk.

[0364] S10. An exosome population, wherein the population comprises any of the exosomes described in the foregoing statements.

[0365] S11. A composition comprising exosomes or groups of exosomes as described in any of the foregoing statements.

[0366] S12. A nutritional composition comprising exosomes or exosome populations according to any one of statements 1 to 10.

[0367] S13. An in vitro method for producing mammalian breast milk exosomes, the method comprising:

[0368] A) Generate mammary gland-like organoids derived from mammalian induced pluripotent stem cells (miPSCs).

[0369] B) The lactating cells secrete mammalian milk-like products, and

[0370] C) Purify the exosomes from the mammalian milk product to remove impurities, optionally by chromatography, filtration, or ultracentrifugation, to separate the exosomes.

[0371] Step A) includes culturing the miPSCs in a culture medium containing BMP4 and / or RA.

[0372] S14. The method according to statement 13, wherein step A) comprises:

[0373] i) Culture the mammalian induced pluripotent stem cells (miPSCs) in a medium containing BMP4 and / or RA to generate embryoid bodies (EBs), and

[0374] ii) Allow the EB to grow to generate a population of mammary cells.

[0375] S15. The method according to statement 14, wherein the culture step i) comprises culturing the miPSCs in a 3D suspension culture system, for example under 3D suspension conditions, in MammoCult medium and BMP4, thereby guiding the iPSCs to differentiate into non-neuroectodermal cells, optionally for at least 12 days.

[0376] S16. The method according to statement 14, wherein the culture step i) comprises culturing the miPSCs in a 3D suspension culture system, for example under 3D suspension conditions, in MammoCult medium and BMP4, thereby guiding the iPSCs to differentiate into non-neural ectodermal cells, optionally for no more than 8 days.

[0377] S17. The method according to statement 14 or 15, wherein the growth step ii) comprises growing the formed EB for at least 30 days, for example 32 days, in a 3D embedding system containing RA, such as in a mixed floating gel composed of matrix proteins such as Matrigel and / or type I collagen, to generate lactating cells.

[0378] S18. The method according to statement 14 or 16, wherein the growth step ii) comprises growing the formed EB for no more than 25 days in a 3D embedding system containing RA, such as in a mixed floating gel composed of matrix proteins such as Matrigel and / or type I collagen, to generate lactating cells.

[0379] S19. The method according to statement 17, wherein step ii) is further divided into sub-steps and includes the following steps: ii) and iii):

[0380] ii) Incubate mEB (mammary globules) for 5 days in complete EpiCult B medium supplemented with EpiCult proliferation supplement, parathyroid hormone (pTHrP) and RA;

[0381] iii) Branching and vesicle differentiation and mammary cell specialization were promoted by incubating the mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 20 days.

[0382] S20. The method according to statement 18, wherein step ii) is further divided into sub-steps and includes the following steps: ii) and iii):

[0383] ii) The formed mEB (mammary globules) were embedded in a mixture of Matrigel and type I collagen floating in EpiCultB medium supplemented with EpiCult proliferative supplement, parathyroid hormone (pTHrP) and RA for 5 days.

[0384] iii) Incubation of embedded mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 15 days promoted branching and vesicle differentiation and mammary cell specialization.

[0385] S21. The method according to any one of statements 13 to 20, wherein the mammary gland cells are human mammary gland cells.

[0386] S22. The method according to any one of statements 13 to 21, wherein BMP4 is added to the culture medium between day 0 and day 10, preferably between day 0 and day 3, wherein day 0 is the time point at which the iPSC is first added to the culture medium.

[0387] S23. The method according to any one of statements 1 to 22, wherein BMP4 is added to the culture medium for 3 days.

[0388] S24. The method according to any one of statements 1 to 23, wherein RA is added to the culture medium between day 10 and day 15, optionally between day 6 and day 11, wherein day 0 is the time point at which the iPSC is first added to the culture medium.

[0389] S25. The method according to any one of statements 1 to 24, wherein RA is added to the culture medium for 5 days.

[0390] S26. The method according to any one of statements 1 to 25, wherein BMP4 is added to the culture medium at a concentration of 5 ng / mL to 20 ng / mL, preferably 5 ng / mL, 10 ng / mL or 20 ng / mL.

[0391] S27. The method according to any one of statements 1 to 26, wherein RA is added to the culture medium at a concentration of 1 µM.

[0392] S28. The method according to any one of statements 1 to 27, wherein the EB expresses one or more mammary gland positive progenitor cell markers, wherein the one or more mammary gland positive progenitor cell markers are optionally selected from EpCAM, CD49f, MUC1 and GATA3.

[0393] S29. The method according to any one of statements 1 to 28, wherein the expression of the breast gland positive progenitor cell markers in the EB is increased compared with the expression level of one or more breast gland positive progenitor cell markers in the EB that has not been treated with BMP4 and RA.

[0394] S30. The method according to any one of statements 1 to 29, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EB express one or more mammary gland positive progenitor cell markers, optionally wherein at least 35% of the EB express EpCAM and CD49f mammary gland positive progenitor cell markers, optionally wherein at least 15% of the EB express MUC1 and EpCAM mammary gland positive progenitor cell markers, and / or optionally wherein at least 20% of the EB express EpCAM and GATA3 mammary gland positive progenitor cell markers.

[0395] S31. The method according to any one of statements 1 to 30, wherein the EB expresses one or more milk-specific bioactive markers, optionally osteopontin (OPN).

[0396] S32. The method according to any one of statements 1 to 31, wherein the expression of one or more milk-specific bioactive markers of the EB is increased, and optionally the expression of osteopontin (OPN) is increased.

[0397] S33. The method according to any one of statements 1 to 32, wherein the secretion of one or more milk-specific bioactive markers of the EB is increased, optionally the secretion of osteopontin (OPN) is increased.

[0398] S34. The method according to any of the foregoing statements, wherein step A) is a process between 30 days and 45 days, optionally less than 45 days, optionally less than 44 days, optionally less than 35 days, optionally less than 31 days.

[0399] S35. The method according to any of the foregoing statements, wherein the method is used to produce a human milk-like product.

[0400] Step A) further includes:

[0401] i) By culturing hiPSCs for at least 12 days in a suitable 3D culture system, such as 3D suspension conditions, in a suitable medium containing BMP4, such as MammoCult medium and BMP4, the hiPSCs are guided to differentiate into non-neural ectodermal cells.

[0402] ii) In a suitable 3D embedding system containing RA, for example, in a mixed floating gel composed of matrix proteins such as Matrigel and / or type I collagen and RA, the formed mEB (mammary globules) are grown for at least 30 days, for example 32 days, to generate lactating cells.

[0403] S36. The method according to any of the foregoing statements, wherein the method is used to produce a human milk substitute product.

[0404] Step A) further includes:

[0405] i) By culturing hiPSCs in a suitable 3D culture system, such as 3D suspension conditions, in a suitable medium containing BMP4, such as MammoCult medium and BMP4, for no more than 8 days, hiPSCs are guided to differentiate into non-neural ectodermal cells.

[0406] ii) Allow the formed mEBs (mammary globules) to grow for no more than 25 days in a suitable 3D embedding system containing RA (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or type I collagen and RA) to generate lactating cells.

[0407] S37. According to the method described in statement 35, step A) i) is defined as follows:

[0408] i) Using standard iPSC medium E8 or mTeSR containing DMEM / F12, magnesium L-ascorbate-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3, and transferrin, TGFβ1, or NODAL. ™ Embryoids (EBs) were generated from hiPSCs by incubation for two days in a medium, and highly enriched mEBs (mammaglobules) of non-neural ectoderm cells were produced by incubating EBs for 10 days in complete MammoCult medium containing basal medium, proliferation supplement, and supplemented with BMP4, heparin, and hydrocortisone.

[0409] Step A)ii) is further divided into sub-steps and includes the following steps ii), iii) and iv):

[0410] ii) Incubate mEB (mammary globules) for 5 days in complete EpiCult B medium supplemented with EpiCult proliferation supplement, parathyroid hormone (pTHrP) and RA;

[0411] iii) Incubation of mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 20 days promoted branching and vesicle differentiation and mammary cell specialization.

[0412] iv) Milk protein expression was induced by incubating mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days.

[0413] S38. According to the method described in statement 36, step A)i) is defined as follows:

[0414] i) Using standard iPSC medium E8 or mTeSR containing DMEM / F12, magnesium L-ascorbate-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3, and transferrin, TGFβ1, or NODAL. ™ Embryoids (EBs) were generated from hiPSCs by incubation for two days in a medium, and highly enriched mEBs (mammaglobules) of non-neural ectoderm cells were produced by incubating EBs for 6 days in a complete MammoCult medium containing basal medium, proliferation supplement, and supplemented with BMP4, heparin, and hydrocortisone.

[0415] Step A)ii) is further divided into sub-steps and includes the following steps ii), iii) and iv):

[0416] ii) Incubate the mEB (mammary globules) for 5 days in complete EpiCult B medium supplemented with EpiCult proliferation supplement, pTHrP and RA;

[0417] iii) Incubation of the mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 15 days promoted branching and vesicle differentiation and mammary cell specialization.

[0418] iv) Milk protein expression was induced by incubating the mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 5 days.

[0419] S39. According to the method described in statement 35, step A) i) is defined as follows:

[0420] i) germ-like bodies (EBs) are generated from hiPSCs by incubating hiPSCs for two days in standard iPSC medium E8 containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenide, FGF2, insulin, NaHCO3, and transferrin, TGFβ1, or NODAL, and highly enriched mEBs (mammaglobules) of non-neuroectoderm cells are generated by incubating the EBs for 10 days in MammoCult B medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin, and BMP4, wherein step A) ii) is further divided into sub-steps and includes the following steps ii), iii), and iv):

[0421] ii) The formed mEB (mammary globules) were embedded in a mixture of Matrigel and type I collagen floating in EpiCultB medium supplemented with EpiCult proliferative supplement, parathyroid hormone (pTHrP) and RA for 5 days.

[0422] iii) Incubation of embedded mEBs (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 20 days promoted branching and vesicle differentiation as well as mammary cell specialization.

[0423] iv) Milk protein expression was induced by incubating mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 7 days.

[0424] S40. The method described in statement 36, wherein step A)i) is defined as follows:

[0425] i) embryoid bodies (EBs) are generated from hiPSCs by incubation for two days in standard iPSC medium E8 containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenide, FGF2, insulin, NaHCO3, and transferrin, TGFβ1, or NODAL, and mEBs (mammaglobules) with highly enriched non-neuroectodermal cells are generated by incubating the EBs for 6 days in MammoCult B medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin, and BMP4, wherein step A) ii) is further divided into sub-steps and includes the following steps ii), iii), and iv):

[0426] ii) The formed mEB (mammary globules) were embedded in a mixture of Matrigel and type I collagen floating in EpiCultB medium supplemented with EpiCult proliferative supplement, parathyroid hormone (pTHrP) and RA for 5 days.

[0427] iii) Incubation of embedded mEBs (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10, and HGF for 15 days promoted branching and vesicle differentiation and mammary cell specialization.

[0428] iv) Milk protein expression was induced by incubating the mEB (mammary globules) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol for 5 days.

[0429] S41. The method according to any of the foregoing statements, wherein step A) is performed under 3D suspension culture conditions.

[0430] S42. 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.

[0431] S43. The method according to any of the foregoing statements, wherein step C) further includes sterilizing the isolated exosomes.

[0432] S44. 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.

[0433] S45. The method according to any of the foregoing statements further includes formulating the isolated exosomes together with supplemental nutritional ingredients.

[0434] S46. The method according to any of the foregoing statements, wherein the isolated exosomes are dispensed into containers for consumption.

[0435] S47. A mammalian breast milk exosome product, said mammalian breast milk exosome product being obtainable by any one of statements 13 to 46, optionally a human breast milk exosome.

[0436] S48. A method for producing mammalian milk fortifier, the method comprising performing any one of statements 13 to 46.

[0437] 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.

[0438] 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

[0439] 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.

[0440] Figure 2The diagram illustrates the differentiation of human induced pluripotent stem cells (hiPSCs) according to step A).

[0441] Figure 3 The illustration shows the differentiation of human induced pluripotent stem cells (hiPSCs) according to preferred and particularly preferred embodiments of the method of the present invention for step A).

[0442] Figure 4 : 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)).

[0443] Figure 5 : 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)).

[0444] Figure 6 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.

[0445] Figure 7This study demonstrates 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. Flow cytometry quantification of the number of mammary gland-positive progenitor cells using several identified markers—EpCAM, CD49f, MUC1, and GATA3—during the differentiation timeline (day 25 (for the control protocol) and day 20 (for the shortened timeline protocol)) and the pre-induction phase (day 35 (for the control protocol including BMP4 and RA) and day 26 (for the shortened timeline protocol)) and the post-induction timeline (day 42 (for the control protocol including BMP4 and RA) and day 31 (for the shortened timeline protocol)) is also shown. *A shortened modified protocol (day 31) including the combination of BMP4 and RA is described.

[0446] Figure 8 : This shows the expression (ah) of different mammary epithelial markers derived from iPSCs in lactating cells in both the normal length and shortened protocols.

[0447] Figure 9 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 controlled differentiation method or a combination of bone morphogenetic protein 4 (BMP4), retinoic acid (RA), and the combination of both.

[0448] Figure 10: a, Venn diagram showing the number of proteins identified only in EVs purified from conditioned medium (days 39 to 41) (blue circles), the number of proteins identified only in EVs purified from human milk (red circles), and the number of proteins identified in both types of EVs (overlapping blue and red 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).

[0449] Figure 11: a, Venn diagram showing the number of miRNAs significantly downregulated in EVs purified from conditioned medium (days 39–41) compared to human milk EVs (blue circles); the number of miRNAs significantly upregulated in conditioned medium EVs compared to human milk EVs (red squares); and the number of miRNAs not expressed differentially in the two types of EVs (overlapping blue and square shapes). b, Table showing a list of the top 4 proteins expressed in both types of EVs (human milk and conditioned medium) (based on FDR).

[0450] Figure 12The 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.

[0451] Figure 13 : Shows the median particle size (nm) of EVs purified from conditioned medium (day 41) and human milk, as analyzed by nanoflow cytometry.

[0452] Experimental Section

[0453] Example 1

[0454] hiPSCs were cultured and differentiated into lactating cells to obtain human milk-like products.

[0455] 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.

[0456] Example 2

[0457] hiPSCs were cultured and differentiated into 3D lactogenic cells to obtain human milk-like products.

[0458] 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.

[0459] Example 3

[0460] Another alternative method is to culture and differentiate hiPSCs into lactating cells to obtain human milk-like products.

[0461] Lactobacillus differentiation can be efficiently obtained from hiPSCs under alternative culture conditions, including conditions 1 through 4 as described below:

[0462] 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).

[0463] 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).

[0464] 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 I collagen), and then cultured for another 25 days in the presence of insulin, HGF, hydrocortisone and FGF10.

[0465] 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.

[0466] Example 4

[0467] 2D and 3D lactocyte differentiation based on human induced pluripotent stem cell (hiPSC) line 603

[0468] (a) 3D lactocyte differentiation based on human induced pluripotent stem cell (hiPSC) line 603:

[0469] 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).

[0470] (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).

[0471] The next day, the culture medium was replaced with E8 (from day -2 to day 0).

[0472] 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.

[0473] (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 three days from day 10 to day 15.)

[0474] (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 three days (from day 15 to day 35).

[0475] (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 three 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... Figure 4 As shown in the image.

[0476] (b) 2D lactocyte differentiation based on human induced pluripotent stem cell (hiPSC) line 603

[0477] 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).

[0478] 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 5 As shown in the image.

[0479] (c) Results

[0480] Using quantitative RT-PCR ( Figure 4 3D differentiation Figure 5 (2D Differentiation) Capturing different differentiation stages during lactation cell derivation. NaNog expression, a marker of pluripotency, was reduced in both 2D and 3D settings, while cells matured and differentiated. 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-neuroelectrodermal 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 only tracked 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.

[0481] 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).

[0482] 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.

[0483] 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 membrane thickness of 100 m × 0.25 mm ID × 0.25 mm 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.

[0484] According to the protocol of Example 4a, the fatty acid results obtained on day 42 are reported in Table 2 (differences were observed between the culture medium and the supernatant).

[0485] Table 2 below lists the fatty acids expressed in cell supernatant samples.

[0486]

[0487] 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.

[0488] Table 3 below lists all the best candidates for cutting the adhesive strip.

[0489]

[0490] 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.

[0491] 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.

[0492] In short, 974 miRNAs were detected in the cell supernatant, of which more than 75 were miRNAs highly expressed in the milk sample.

[0493] Table 4 below lists the ten miRNAs with the highest expression levels.

[0494]

[0495] 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.

[0496] Example 5

[0497] 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 a biomimetic model of human mammary glands. These methods were implemented over 42 days and 31 days (as a shortened time series). Figure 6 ).

[0498] 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 7 ac) and post-induction phase ( Figure 7 During the df period, their expression profiles are maintained.

[0499] We evaluated the expression of different mammary epithelial markers in lactating cells derived from iPSCs in both normal length and shortened protocols. Figure 8 This shortened protocol induces RELA as a specific gene used to identify mammary epithelial cell lineages during iPSC differentiation. Figure 8 a). In the shortened scheme ( Figure 8 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 8 f). Similarly, shortened differentiation process is associated with elevated levels of mammary gland progenitor cell markers such as CD24 and ITGA6 (CD49f). Figure 8 gh).

[0500] Human osteopontin peptides -GDSVVYGLR and -YPDAVATWLNPDPSQK were specifically detected in cell culture supernatant by LC-MS / MS. Figure 9 ).

[0501] Example 6

[0502] 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.

[0503] 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).

[0504] 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.

[0505] 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 10a ). Figure 10b The 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.

[0506] 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 11a This includes the detection of validated miRNAs such as miR-148a-3p, miR-22-3p, miR-125b-5b, and miR-6126. Figure 11b ).

[0507] 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 12 The same phenomenon was observed when analyzing subclass spectra, such as phospholipid spectra.

[0508] Flow cytometry analysis of isolated EVs from conditioned medium (step A) or breast milk control showed that their size range was similar, from 65 nm to 75 nm.

[0509] 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, wherein the exosomes do 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).

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

3. The exosome according to claim 1 or 2, wherein the exosome comprises one or more miRNAs selected from miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

4. 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.

5. 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.

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

7. The exosomes according to claim 6, wherein the diameter of the exosomes is 65 nm to 75 nm.

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

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

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

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

12. An in vitro method for producing exosomes or exosome populations according to any one of claims 1 to 11, the method comprising: A) Generate mammary gland-like organoids derived from human induced pluripotent stem cells (hiPSCs). B) The lactating cells secrete human milk-like products, and C) Purify the exosomes from the human milk-like product to remove impurities, optionally by chromatography, filtration, or ultracentrifugation, to separate the exosomes. Step A) includes culturing the hiPSCs in a culture medium containing BMP4 and / or RA.

13. The method of claim 12, wherein step A) is a process between 30 days and 45 days.

14. The method according to claim 12 or 13, wherein step A) is performed under 3D suspension culture conditions.

15. The method according to claims 12 to 14, 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.

16. The method according to claims 12 to 15, wherein step C) further comprises sterilizing the isolated exosomes.

17. The method according to claims 12 to 16, wherein step C) further comprises storing the isolated exosomes at a temperature below freezing, optionally at -80°C.

18. The method according to claims 12 to 17, further comprising formulating the isolated exosomes together with supplemental nutritional ingredients.

19. The method according to claims 12 to 18, wherein the separated exosomes are dispensed into containers for consumption.

20. A human breast milk exosome product, said human breast milk exosome product being obtainable by the method according to any one of claims 12 to 19.

21. A method for producing a human milk fortifier, the method comprising performing the method according to any one of claims 12 to 19.

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