Method for producing a milk-like product

Abstract

Methods for producing mammary cells, and methods for producing a mammalian milk-like product, such as a human milk-like product, comprising generating lactocytes derived from mammalian induced pluripotent stem cells (miPSCs), such as human induced pluripotent stem cells (hiPSCs), and expressing the mammalian milk-like product, such as the human milk-like product, from the lactocytes.

Description

[Technical field]

[0001] The present invention relates to a method for producing mammary gland cells. The present invention also relates to a method for in vitro production of mammalian milk-like products, such as human milk-like products, comprising generating lactocytes derived from mammalian induced pluripotent stem cells (hiPSCs), such as human induced pluripotent stem cells (hiPSCs), by culture and differentiation, and / or generating mammary gland-like organoids comprising such lactocytes, and expressing mammalian milk-like products, such as human milk-like products, from such lactocytes and / or mammary gland-like organoids. The present invention also relates to mammalian milk-like products, such as human milk-like products, that can be obtained from such methods.

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

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

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

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

[0006] It is therefore an object of the present invention to provide improved methods for producing mammary gland cells and to reproduce the expression of mammalian milk (e.g., human milk) in cultured cells. It is also an object of the present invention to prepare customized mammalian milk-like products, e.g., human milk-like products, in cultured cells that can be adapted to the specific needs of the recipient and / or to produce human milk bioactives to complement existing bovine-based solutions for infant nutrition.

[0007] [Summary of the invention] The present invention solves the above technical problems. Provided herein is a method for producing a population of mammary gland cells, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium containing bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) to generate embryoid bodies (EBs); ii) growing the EBs to generate a population of mammary cells.

[0008] Also provided herein is the use of BMP4 and RA to improve the efficiency of differentiation of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells in a differentiation protocol.

[0009] Further provided herein is a method of producing a mammalian milk-like product, comprising: A) generating lactocytic mammary-like gland organoids derived from mammalian induced pluripotent stem cells (miPSCs); B) secreting a mammalian milk-like product from said lactocytes derived from mammalian induced pluripotent stem cells (miPSCs), The method, wherein step A) comprises culturing the miPSCs in a culture medium containing BMP4 and RA.

[0010] Further provided herein is a human milk-like product obtainable according to the methods described herein.

[0011] Further provided herein is a human milk-like product as described herein for use in therapy.

[0012] Finally, there is provided herein the use of the human breast milk-like products described herein as human milk substitutes, optionally as breast milk substitutes.

[0013] [Mode for carrying out the invention] definition In the context of the present invention, the term "in vitro" means performed or occurring in a test tube, culture dish, bioreactor, or anywhere outside the body of a living organism.

[0014] In the context of the present invention, the term "mammal" refers to animals belonging to the mammalian species, such as humans, cows, monkeys, camels, sheep, goats, etc.

[0015] In the context of the present invention, the term "lactocytes" or "mammary gland-like cells" identifies secretory epithelial cells that express the CK18 cell marker and are derived from mammalian induced pluripotent stem cells (miPSCs), in particular human induced pluripotent stem cells (hiPSCs). Human induced pluripotent stem cells (hiPSCs) as used herein are commercially available and can be selected from any suitable hiPSC line. A suitable human induced pluripotent stem cell line in the context of the present invention is, for example, the hiPSC line 603 commercially available from Fujifilm Cellular Dynamics, Inc. (FCDI), which is used according to the present invention. Further suitable hiPSCs can be selected, for example, as described in Ying Qu et al., (2017, supra). In one embodiment of the present invention, the hiPSCs are not engineered. In one embodiment, the hiPSCs are not engineered to include an exogenous nucleic acid and / or an inducible gene expression system comprising an exogenous nucleic acid. The inducible gene expression system is configured to express a hormone or a signaling factor. In one embodiment, the exogenous nucleic acid and / or an inducible gene expression system comprising the exogenous nucleic acid promotes cell differentiation into lactocytes.

[0016] In the context of the present invention, the term "mammary gland-like organoid" or "mammary-like organoid" refers to a miniaturized and simplified version of the mammary gland, developed in two or three dimensions (2D / 3D) and containing lactocytes as defined above.

[0017] In the context of the present invention, the term "human milk-like product" refers to a cell-cultured milk product. Such a product is an edible product expressed by the lactocytes and / or mammary gland-like organoids produced according to the methods of the present invention.

[0018] The "human milk-like product" according to the present invention can have the same components (e.g., in terms of bioactives, macronutrients and micronutrients and their levels) as human breast milk from a well-nourished mother, which is referred to herein as a "standardized human milk product". Alternatively, the "human milk-like product" according to the present invention can have variations in the ratios and concentrations of components naturally found in human breast milk from a well-nourished mother, which is referred to herein as a "non-standardized milk-like product". The "human milk-like product" according to the present invention can be modified to include components not naturally found in human breast milk from a well-nourished mother ("modified milk-like product"). Non-limiting examples of human milk-like products are selected from the group consisting of supplements, fortifiers, human breast milk substitutes (or replacements), and ingredients enriched in only one and / or some of the bioactives, macronutrients and micronutrients that can typically be found in human breast milk from a well-nourished mother.

[0019] "Human milk-like products" can be used as an alternative to ingesting naturally secreted milk ("human milk substitutes"). Milk substitute products can be used as supplements ("human milk supplements") or fortifiers ("human milk fortifiers") to be ingested in combination with naturally secreted milk.

[0020] In one embodiment, the standard human milk-like product according to the invention comprises at least the macronutrients and micronutrients that can typically be found in human breast milk from well-nourished mothers. In one embodiment, the human milk-like product according to the invention comprises proteins, peptides, lipids (including linoleic acid and alpha-linolenic acid), carbohydrates, vitamins (including vitamin A, vitamin D3, vitamin E, vitamin K, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, pantothenic acid, folic acid, vitamin C and biotin), minerals (including iron, calcium, phosphorus, magnesium, sodium, chloride, potassium, manganese, iodine, selenium, copper and zinc), choline, myo-inositol and L-carnitine. In one embodiment, the human milk-like product according to the invention also comprises at least one bioactive substance selected from the group consisting of growth / growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and / or exosomes), exosome-derived bioactive substances (e.g. miRNA) and secretory IgA. The standard human milk-like product according to the present invention is not a naturally occurring human breast milk secretion.

[0021] In another embodiment, the human milk-like product according to the invention can be adapted to the specific needs of the infant consuming such product. The human milk-like product according to the invention can contain only one and / or some of the bioactive substances, macronutrients and micronutrients that can typically be found in human breast milk from a well-nourished mother. In such an embodiment, the human breast milk-like product may also be referred to by the term "non-standard human milk-like product". In one embodiment, a non-standard human milk-like product according to the invention comprises one or more nutrients or bioactives selected from the group consisting of proteins, peptides, lipids (including linoleic acid and alpha-linolenic acid), carbohydrates (including human milk oligosaccharides), vitamins (including vitamin A, vitamin D3, vitamin E, vitamin K, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, pantothenic acid, folic acid, vitamin C and biotin), minerals (including iron, calcium, phosphorus, magnesium, sodium, chloride, potassium, manganese, iodine, selenium, copper and zinc), choline, myo-inositol, L-carnitine, growth / growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and / or exosomes), exosome-derived bioactives (e.g. miRNA) and secretory IgA.

[0022] In the context of the present invention, the term "unmodified human milk-like product" refers to the human milk-like product expressed by lactocytes and / or by the mammary gland-like organoids produced according to step A) and step B) of the method of the present invention, and not subjected to further processing according to optional step C) of the method of the present invention. Unmodified human milk-like product can include both standard and non-standard human milk-like products. Non-limiting examples of non-standard human milk-like products are selected from the group consisting of supplements, fortifiers, and raw materials enriched with only one and / or some of the bioactive substances, macronutrients and micronutrients that can be typically found in human breast milk from well-nourished mothers.

[0023] In the context of the present invention, the term "modified human milk-like product" refers to a human milk-like product expressed by lactocytes and / or by mammary gland-like organoids generated according to steps A) and B) of the method of the present invention and subjected to further processing according to optional step C) of the method of the present invention.

[0024] Modified human milk-like products can include both standard and non-standard human milk-like products.

[0025] In the context of the present invention, the term "EB" means "embryoid body."

[0026] In the context of the present invention, the term "mEB" means "embryoid bodies cultured in MammoCult medium."

[0027] MammoCult Medium refers to a serum-free culture medium that contains basal medium, at least one growth supplement, heparin and hydrocortisone.

[0028] In the context of the present invention, the terms "embryoid bodies (eBS)", "embryoid bodies cultured in MammoCult medium (mEBs)", "mammospheres" and / or "spheroids" refer to three-dimensional aggregates formed in suspension by pluripotent stem cells (PSCs) under step A) of the method of the present invention.

[0029] The term "infant" in the context of the present invention refers to a child under 12 months of age, such as under 9 months of age, in particular under 6 months of age.

[0030] In the context of the present invention, an infant may be either a full-term infant or a preterm infant. In one embodiment of the present invention, the infant is selected from the group of preterm infants and full-term infants.

[0031] The term "term infant" refers to an infant born at full term or at a gestational age of 37 weeks or greater.

[0032] The term "preterm infant" refers to an infant born at a gestational age less than 37 weeks.

[0033] In the context of the present invention, the term "birth weight" refers to the initial weight acquired by a fetus or newborn after birth.

[0034] In the context of the present invention, the term "low birth weight" means a birth weight below 2500 g (2499 g or less).

[0035] In the context of the present invention, the term "very low birth weight" means a birth weight of less than 1500 g (1499 g or less).

[0036] In the context of the present invention, the term "very low birth weight" means a birth weight of less than 1000 g (999 g or less).

[0037] The term "small-for-gestational-age infants" refers to infants whose birth weight is more than 2 standard deviations below the mean reference for birth weight on the gestational growth chart or 10 standard deviations below the mean reference for birth weight on the gestational growth chart. th Refers to infants who have a birth weight below the percentile. The term "small for gestational age" includes infants who are small at birth, either from constitutive or genetic origins, or as a result of intrauterine growth restriction.

[0038] In the context of the present invention, the term "young children" or "toddlers" refers to children aged between 1 and 3 years.

[0039] The term "infant formula" as used herein refers to a nutritional composition intended for infants and as defined in Codex Alimentarius, (Codex STAN 72-1981) and foods for special dietary uses for infants (including foods for special medical purposes) as defined in Codex Alimentarius, (Codex STAN 72-1981). Infant formula also refers to a food intended for specific nutritional use in infants during the first month of life, which by itself meets the nutritional requirements of infants falling within this category (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant and follow-on formulas). Infant formula includes starter infant formulas and follow-up or follow-on formulas. Generally, starter formulas are breast milk substitutes for infants from birth, and follow-up or follow-on formulas from six months onwards.

[0040] "Growing up milk" (or GUM) is given from the first year onwards. This milk is generally a milk-based drink that is specifically tailored to the nutritional needs of children. Such drinks are nutritional compositions that are used in combination with other foods to feed children from 12 months to 2-3 years of age.

[0041] In the context of the present invention, the term "fortifier" refers to a composition that contains one or more nutrients that have a nutritional benefit to an infant or young child.

[0042] The term "milk fortifier" refers to any composition used to fortify or supplement either human breast milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients. Thus, the human milk fortifier of the present invention can be administered after dissolution in human breast milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients, or it can be administered as a separate composition.

[0043] When administered as a separate composition, the human milk fortifier of the present invention may also be identified as a "supplement." In one embodiment, the human milk fortifier of the present invention is a supplement.

[0044] The term "human milk fortifier" refers to any composition used to fortify or supplement human breast milk, or human breast milk fortified with other nutrients. A "human milk fortifier" according to the present invention may be intended to be administered to infants born prematurely with very low birth weight (VLBW) or extremely low birth weight (ELBW).

[0045] The milk fortifier according to the present invention may be a powder in liquid form.

[0046] Milk fortifier compositions having a liquid form offer some particular advantages: for example, liquid formulations may be more convenient when combined with packaging that delivers calibrated drops of a specific weight or volume.

[0047] Furthermore, liquid formulations are easier to mix with the composition to be fortified, while powder formulations may sometimes form lumps.

[0048] In the context of the present invention, the term "improving the differentiation efficiency and maturation of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells" means increasing the proportion of mammary progenitor cells compared to non-mammary progenitor cells generated from a starting population of miPSCs via a differentiation protocol. In the present invention, the differentiation protocol includes the use of BMP4 and RA. Thus, the increase in the proportion of mammary progenitor cells can be compared to the proportion of mammary progenitor cells to non-mammary progenitor cells generated from a starting population of hiPSCs via an otherwise identical differentiation protocol that does not include the use of BMP4 and RA.

[0049] In the context of the present invention, the term "improving viability" means increasing the number of live and healthy cells, which are capable of a further differentiation step, for example, in the context of the present invention, capable of differentiation into mammary organoids.

[0050] In the context of the present invention, the term "mammary progenitor cell" or similar refers to a cell that expresses at least two mammary progenitor markers, including, but not limited to, CD49f, EpCAM, MUC1, and GATA3. Conversely, in the context of the present invention, the term "non-mammary progenitor cell" or similar refers to a cell that does not express at least two of the mammary progenitor markers.

[0051] References herein to EpiCult Medium or EpiCultB Medium refer to serum-free culture medium containing hydrocortisone, insulin, FGF10 and HGF.

[0052] Culture media as disclosed herein refers to a solid, semi-solid or liquid containing essential nutrients designed to support the growth and differentiation of microorganisms. MammoCult medium is one example of a culture medium that may be used in the present invention.

[0053] Methods and uses according to the present invention The present invention relates to methods for producing mammary gland cells using iPSCs cultured and differentiated under specific conditions, and to methods for using the mammary gland cells in methods for the in vitro production of mammalian milk-like products.

[0054] Surprisingly, it has been demonstrated herein that the addition of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) in the methods described herein increases the efficacy of the differentiation protocol and improves the yield and quality of the final milk-like product. More specifically, it has been demonstrated that BMP4 and RA improve the differentiation efficiency of iPSCs into mammary progenitor cells, and thus increase the number of mammary progenitor cells produced by the methods described herein. The benefits include a higher yield of mammary cells. It has been demonstrated that BMP4 and RA improve the expression and secretion levels of milk-specific bioactive markers, such as osteopontin (OPN). It has also been demonstrated that the length of the overall differentiation protocol can be shortened due to the improved differentiation efficiency by the combination of BMP4 and RA. This shortening has significant advantages, including reduced cell death, higher yield of milk-like material, and cost savings.

[0055] Manufacture of mammalian mammary cells In one aspect, the invention relates to a method for producing mammary gland cells using iPSCs cultured and differentiated under specific conditions.

[0056] Accordingly, the present invention provides a method for producing a population of mammary gland cells, comprising the steps of: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium containing bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) to generate embryoid bodies (EBs); ii) growing the EBs to generate a population of mammary cells.

[0057] The present invention also provides the use of BMP4 and RA to improve the efficiency of differentiation of mammalian induced pluripotent stem cells (mhiPSCs) into mammary progenitor cells in a differentiation protocol. In some embodiments, the present invention also provides the use of BMP4 and RA to improve the efficiency of production of a mammalian milk-like product.

[0058] In some embodiments, mammary gland cells are human mammary gland cells.In some embodiments, mammary gland cells form lactocytic mammary gland-like gland organoids.Such lactocytic mammary gland-like gland organoids are lactogenic, i.e. can produce milk.

[0059] BMP4 is added to the culture medium at an early stage of the differentiation methods described herein. In some embodiments, BMP4 is added to the culture medium between days 0 and 10. In some embodiments, BMP4 is added to the culture medium between days 0 and 6. In some embodiments, BMP4 is added to the culture medium between days 0 and 3. Day 0 is the time when iPSCs are first added to the culture medium, i.e., the time when iPSCs are first induced to differentiate.

[0060] In some embodiments, BMP4 is added to the culture medium for 3 days.

[0061] In some embodiments, BMP4 is added to the culture medium at a concentration of 5 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.

[0062] In some embodiments, BMP4 is added to the culture medium on days 0 to 3 at a concentration of 5-20 ng / mL.

[0063] RA is added to the culture conditions in the early stages of the differentiation methods described herein. In some embodiments, RA is added to the culture medium during the mammary lineage commitment stage. In some embodiments, RA is added to the culture medium between days 10 and 15. In some embodiments, RA is added to the culture medium between days 6 and 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 when iPSCs are first added to the culture medium, i.e., the time when iPSCs are first induced to differentiate.

[0064] In some embodiments, RA is added to the culture medium for 5 days.

[0065] In some embodiments, RA is added to the culture medium at a concentration of 1 uM.

[0066] In some embodiments, RA is added to the culture conditions at a concentration of between 1 μM between days 10 and 15.

[0067] In some embodiments, BMP4 is added to the culture medium between days 0-3, and RA is added to the culture medium between days 10-15.

[0068] In some embodiments, BMP4 is added to the culture medium between days 0-3, and RA is added to the culture medium between days 6-11.

[0069] The EBs generated in the methods described herein express one or more mammary positive progenitor cell markers, hi some embodiments, the one or more mammary positive progenitor cell markers are selected from EpCAM, CD49f, MUC1, and GATA3.

[0070] In some embodiments, expression of one or more mammary positive progenitor cell markers in EBs is increased compared to the expression level of the mammary positive progenitor cell marker in EBs that are not treated with BMP4.

[0071] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more of the EBs express one or more mammary positive progenitor cell markers. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more of the EBs express two or more mammary positive progenitor cell markers. In some embodiments, the mammary positive progenitor cell markers are selected from EpCAM, CD49f, MUC1, and GATA3.

[0072] In some embodiments, at least 35% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, at least 60% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers at an intermediate differentiation stage. In some embodiments, at least 35% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers at a pre-induction stage. In some embodiments, at least 15% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers at a post-induction stage. In some embodiments, at least 40% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers at a post-induction stage.

[0073] In some embodiments, at least 15% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, at least 20% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers at an intermediate differentiation stage. In some embodiments, at least 15% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers at a pre-induction stage. In some embodiments, at least 5% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers at a post-induction stage. In some embodiments, at least 15% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers at a post-induction stage.

[0074] In some embodiments, at least 20% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers. In some embodiments, at least 50% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers at an intermediate differentiation stage. In some embodiments, at least 25% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers at a pre-induction stage. In some embodiments, at least 15% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers at a post-induction stage. In some embodiments, at least 40% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers at a post-induction stage.

[0075] In some embodiments, the intermediate differentiation stage is during day 25, the pre-induction stage is during day 35, and the post-induction stage is during day 42. In some embodiments, the intermediate differentiation stage is during day 20. The pre-induction stage is during day 26, and the post-induction stage is during day 31.

[0076] In some embodiments, the EBs express one or more milk-specific bioactive markers, hi some embodiments, the milk-specific bioactive marker is osteopontin (OPN).

[0077] In some embodiments, the expression of one or more milk-specific bioactive markers in the EBs is increased compared to the expression levels of the markers in EBs not treated with BMP4 and RA. In some embodiments, the secretion of one or more milk-specific bioactive markers in the EBs is increased compared to the expression levels of the markers in EBs not treated with BMP4 and RA. In some embodiments, the secretion of OPN in the EBs is increased compared to the expression levels of OPN in EBs not treated with BMP4 and RA. In some embodiments, the secretion of OPN in the EBs is increased by 40% or more compared to the expression levels of OPN in EBs not treated with BMP4 and RA.

[0078] The methods of producing a population of mammary gland cells as described herein may be combined with and / or utilized in a method for producing a mammalian milk-like product as described herein, particularly as part of step A) as described herein.

[0079] For example, in some embodiments of the methods of producing a population of mammary cells described herein, the culturing step i) comprises culturing miPSCs in a three-dimensional suspension culture system, e.g., in three-dimensional suspension conditions, in MammoCult medium and BMP4, thereby inducing differentiation of the iPSCs into non-neural ectodermal cells. In some embodiments, step Ai) is for at least 12 days. In some embodiments, step Ai) is for 8 days or less.

[0080] In some embodiments of the methods of producing a population of mammary gland cells described herein, growing step ii) comprises growing EBs in a three-dimensional embedding system comprising RA, e.g., a mixed suspension gel composed of matrix proteins such as Matrigel and / or collagen I. In some embodiments, step Aii) is for at least 30 days, e.g., 32 days. In some embodiments, step Aii) is for at least 23 days, e.g., 25 days or less.

[0081] Production of mammalian milk-like products In another aspect, the present invention relates to a method for producing a mammalian milk-like product as defined herein, comprising any of steps A) and B) as defined herein, and any step C) as defined herein. The method for producing a mammalian milk-like product as defined herein may also comprise, as part of step A), a method for producing a mammalian mammary gland cell. In particular, BMP4 and RA may be added to any of the methods for producing a mammalian milk-like product as defined herein, in particular as part of step A). ​​The method may also comprise different lengths of time for producing a mammalian milk-like product.

[0082] Step A - Generation of lactocytes and / or mammary-like organoids from hiPSCs According to the method of the present invention, mammary gland-like cells and / or organoid structures are generated under step A).

[0083] Such mammary-like cells and / or organoid structures can be generated according to any reported method utilizing iPSCs.

[0084] In one embodiment, such mammary gland-like cells and / or organoid structures can be produced according to the procedures described in Ying Qu et al., Stem Cell Reports, Vol. 8, 205-215, which is incorporated herein in its entirety.

[0085] More precisely, the methodology described in the above mentioned scientific publication (hereinafter also referred to as "Ying Qu's publication" or Ying Qu et al. (2017)) represents a two-step protocol for generating human mammary-like cells and / or organoids from iPSCs.

[0086] The protocol preferably includes, as a first step (step 1), differentiation and enrichment of non-neurectodermal cell-containing spheres (mEBs / mammospheres) from iPSCs, and, as a second step (step 2), generation of mammary-like organoids from day 10 mEBs (mammospheres) using three-dimensional floating mixed gel culture of Matrigel and collagen I.

[0087] More specifically, step A preferably comprises the steps of: Embryoid body formation, Mammary lineage fate determination, Branching and acinar differentiation, and Induction of milk bioactive substances.

[0088] Each of these steps may be carried out using a particular culture medium and for a particular period of time.

[0089] In some embodiments, step A is carried out for a total of 40-45 days, preferably 42 days.

[0090] In some embodiments, step A is shortened and occurs for less than 42 days, optionally less than 40 days, optionally less than 31 days. Preferably, step A occurs for 31 days.

[0091] In some embodiments, the embryoid body formation stage is between days 0 and 10. In some embodiments, the embryoid body formation stage is for 10 days.

[0092] In some embodiments, the embryoid body formation stage is shortened and is between days 0 and 6. In some embodiments, the embryoid body formation stage is 6 days. In some embodiments, the embryoid body formation stage is 10 days or less.

[0093] In some embodiments, the mammary lineage commitment stage is between day 10 and day 15. In some embodiments, the mammary lineage commitment stage is between day 6 and day 11. In some embodiments, the embryoid body formation stage is 5 days. In some embodiments, the embryoid body formation stage is 5 days or less.

[0094] In some embodiments, the branching and acinar differentiation stages are between days 15 and 35. In some embodiments, the branching and acinar differentiation stages are for 20 days.

[0095] In some embodiments, the branching and acinar differentiation stages are shortened and are between days 11 and 26. In some embodiments, the branching and acinar differentiation stages are 15 days. In some embodiments, the branching and acinar differentiation stages are 15 days or less.

[0096] In some embodiments, the milk bioactive induction phase is between days 35 and 42. In some embodiments, the milk bioactive induction phase is 7 days.

[0097] In some embodiments, the milk bioactive induction phase is shortened and is between days 26 and 31. In some embodiments, the milk bioactive induction phase is 5 days. In some embodiments, the milk bioactive induction phase is 5 days or less.

[0098] Thus, in some embodiments, step A is carried out for a total of 42 days, wherein the embryoid body formation stage is between days 0 and 10, the mammary lineage commitment stage is between days 10 and 15, the branching and acinar differentiation stage is between days 15 and 35, and the milk bioactive induction stage is between days 35 and 42.

[0099] In other embodiments, step A is carried out for a total of 31 days, where the process is shortened such that the embryoid body formation stage is between days 0 and 6, the mammary lineage fate commitment stage is between days 6 and 11, the branching and acinar differentiation stage is between days 11 and 26, and the milk bioactivity induction stage is between days 26 and 31.

[0100] Further details of time periods and culture media for use in the methods described herein are provided below.

[0101] In step 1, differentiation and enrichment of non-neurectodermal cell-containing spheres (mEBs / mammospheres) from hiPSCs is performed by culturing hiPSCs in complete MammoCult medium (StemCell Technologies). Complete MammoCult medium is preferably composed of basal medium, growth supplements, heparin (typically 4 μg / mL), and hydrocortisone (typically 0.48 μg / mL). The medium is usually changed every 3 days. The mEBs (mammospheres) obtained in the previous step are then enriched for non-neurectodermal cells.

[0102] In some embodiments, BMP4 is added to the culture medium in step 1.

[0103] BMP4 is added to the culture medium as described herein.

[0104] BMP4 is added to the culture conditions at an early stage of the differentiation method as described herein. In some embodiments, BMP4 is added to the culture medium between days 0 and 10. In some embodiments, BMP4 is added to the culture medium between days 0 and 6. In some embodiments, BMP4 is added to the culture medium between days 0 and 3. Day 0 is the time when iPSCs are first added to the culture medium, i.e., the time when iPSCs are first induced to differentiate.

[0105] In some embodiments, BMP4 is added to the culture medium for 3 days.

[0106] In some embodiments, BMP4 is added to the culture medium at a concentration of 5 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.

[0107] In some embodiments, BMP4 is added to the culture medium between days 0 and 3 at a concentration of 5-20 ng / mL.

[0108] In step 2, three-dimensional cultures are first prepared based on floating mixed gels (e.g., Matrigel and Collagen I) to generate mammary-like organoids according to the protocol of Ying Qu et al. (2017). Then, day 10 mEBs (mammospheres) are grown for 5 days in the floating mixed gels in complete EpiCultB medium supplemented with parathyroid hormone (pTHrP). The cells are then cultured in complete EpiCultB medium supplemented with hydrocortisone and insulin and FGF10 and HGF for induction of branching and acinar differentiation for the preparation of mammary-like organoids / lactocytes. Milk protein expression is typically induced on day 35 by culturing in complete EpiCultB medium supplemented with BSA (milk secretion medium) with the addition of prolactin, hydrocortisone and insulin for 5 days. The process of Ying Qu et al. (2017) is typically completed on day 40.

[0109] In some embodiments, RA is added to the culture medium in step 2.

[0110] RA is added to the culture medium as described herein.

[0111] RA is added to the culture conditions at an early stage of the differentiation methods described herein. In some embodiments, RA is added to the culture medium during the mammary lineage commitment stage. In some embodiments, RA is added to the culture medium between days 10 and 15. In some embodiments, RA is added to the culture medium between days 6 and 11. In some embodiments, RA is not added to the culture medium after day 11. Day 0 is the time when iPSCs are first added to the culture medium, i.e., the time when iPSCs are first induced to differentiate.

[0112] In some embodiments, RA is added to the culture medium for 5 days.

[0113] In some embodiments, RA is added to the culture medium at a concentration of 1 μM.

[0114] In some embodiments, RA is added to the culture medium at a concentration of between 1 μM between days 10 and 15.

[0115] In one embodiment of the present invention, there is provided a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating iPSCs into non-neurectodermal cells by culturing the iPSCs in an appropriate culture medium as described herein (e.g., MammoCult Medium) and BMP4, and harvesting mammospheres formed from the cells after 10 days; and ii) growing such mammospheres for at least 10 days in a suitable system containing RA as described herein (e.g., the floating mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012) to generate lactocytes.

[0116] In one embodiment of the present invention, there is provided a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating the iPSCs into non-neuroectodermal cells by culturing the iPSCs in an appropriate culture medium (e.g., MammoCult medium) and harvesting the formed mammospheres after 6 days of BMP4 as described herein; and ii) growing such mammospheres in a suitable system containing RA as described herein (e.g., the floating mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012) for less than 10 days to generate lactocytes.

[0117] In another embodiment, there is provided a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating iPSCs into non-neurectodermal cells by culturing iPSCs in a suitable culture medium as described herein (e.g., MammoCult Medium) and BMP4 in non-adherent conditions for mammosphere formation; and ii) growing such mammospheres in a suitable three-dimensional system (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen, or a suspension culture in a non-adherent plate) containing RA as described anywhere herein for at least 10 days to generate lactocytes.

[0118] In another embodiment, there is provided a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating iPSCs into non-neurectodermal cells by culturing iPSCs in a suitable culture medium as described herein (e.g., MammoCult Medium) and BMP4 in non-adherent conditions for mammosphere formation; and ii) growing such mammospheres in a suitable three-dimensional system (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen, or a suspension culture in a non-adherent plate) containing RA as described anywhere herein for less than 10 days to generate lactocytes.

[0119] In one embodiment, mammary gland fate commitment under step A) is obtained by applying a conditioned medium (e.g., EpiCultB) supplemented with specific factors (e.g., parathyroid hormone (pTHrP), hydrocortisone, insulin, FGF10, and HGF) and RA.

[0120] In one embodiment of the present invention, the method comprises generating mammary-like organoids under step A).

[0121] In one embodiment of the present invention, the method for generating mammary-like organoids under step A) comprises culturing cells under conditions selected from the group consisting of 2D monolayer of cells, 2D with attached EBs, suspension in non-adherent plates, and mixed floating gel.

[0122] In a preferred embodiment, the mixed floating gel comprises Matrigel and Collagen I.

[0123] In another preferred embodiment, the mammospheres (mEBs) in step A) are grown for at least 15 days in a suitable system (e.g., the suspension mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012).

[0124] In another more preferred embodiment, the mammospheres (mEBs) in step A) are grown for 20 days in an appropriate system (eg, the suspension mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012).

[0125] In one embodiment, the method according to the invention provides culture conditions according to step A) [eg step A) i) and / or ii)].

[0126] The culture conditions are adjusted to generate lactocytes derived from human induced pluripotent stem cells (hiPSCs) capable of secreting human milk-like products.

[0127] In a preferred embodiment, a method for producing a human milk-like product is provided, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising inducing differentiation of hiPSCs into mammary cells (e.g., mammary cells) in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) comprising BMP4 and RA as described herein. In some embodiments, for at least 42 days. In some embodiments, for no more than 31 days. In some embodiments, for at least 31 days.

[0128] In another preferred embodiment, there is provided a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating the hiPSCs into non-neurectodermal cells by culturing the hiPSCs in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) in a suitable culture medium (e.g., MammoCult medium) and BMP4 as described herein for at least 12 days (day -2 to day 10); and ii) growing the formed mEBs (mammospheres) for at least 30 days, preferably 32 days, in a suitable three-dimensional embedding system (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen I) containing the RA described herein to generate lactocytes.

[0129] In another preferred embodiment, there is provided a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating the hiPSCs into non-neurectodermal cells by culturing the hiPSCs in a suitable culture medium (e.g., MammoCult medium) and BMP4 as described herein in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) for up to 8 days (day -2 to day 6); and ii) growing the formed mEBs (mammospheres) in an appropriate three-dimensional embedding system (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen I) containing RA as described herein for up to 25 days to generate lactocytes.

[0130] In a particularly preferred embodiment of the present invention, a method for producing a human milk-like product is provided, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), wherein step A)i) is defined as follows: i) generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days (day -2 to day 0) in standard iPSC medium E8 (containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL as described in Chen et al., Nat Methods, 2011) or mTeSR™, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 10 days (day 0 to day 10) in complete MammoCult medium (StemCell Technologies) containing basal medium, growth supplements and supplemented with heparin (typically 4 μg / mL), hydrocortisone (typically 0.48 μg / mL) and BMP4 as described herein; Step A) ii) is differentiated into further sub-steps and the following steps: ii), iii) and iv): ii) incubation of mEBs (mammospheres) for 5 days (days 10-15) in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP); iii) promotion of branching and acinar differentiation and mammary cell specification by incubation of mEBs (mammospheres) for 20 days (days 15-35) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF; and iv) Induction of milk protein expression by incubating mEBs (mammospheres) for 7 days (days 35-42) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0131] In a particularly preferred embodiment of the present invention, a method for producing a human milk-like product is provided, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), wherein step A)i) is defined as follows: i) generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days (day -2 to day 0) in standard iPSC medium E8 (containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3, and transferrin, TGFβ1 or NODAL as described in Chen et al., Nat Methods, 2011) or mTeSR™, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 6 days (day 0 to day 6) in complete MammoCult medium (StemCell Technologies) containing basal medium, growth supplements, and supplemented with heparin (typically 4 μg / mL), hydrocortisone (typically 0.48 μg / mL), and BMP4 as described herein, where Step A) ii) is differentiated into further sub-steps and the following steps: ii), iii) and iv): ii) incubation of mEBs (mammospheres) for 5 days (days 6-11) in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP) and RA; iii) promoting branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) for 15 days (days 11-26) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF; and iv) Induction of milk protein expression by incubating mEBs (mammospheres) for 5 days (days 26-31) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0132] Step iv) preferably results in differentiation into milk protein expressing cells, in particular lactocytes, and / or mammary gland-like organoids.

[0133] In a further particularly preferred embodiment of the present invention, a method for producing a human milk-like product is provided, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), wherein step A)i) is defined as follows: i) generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days (day 2-day 0) in standard iPSC medium E8 (containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL as described in Chen et al., Nat Methods, 2011) or mTeSR™; and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 10 days (day 0-day 10) in MammoCultB medium supplemented with MammoCult growth supplements, hydrocortisone, heparin and BMP4 as described herein, where step A)ii) is differentiated into further sub-steps and the following steps ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I suspended in EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP) and RA for 5 days (days 10-15); iii) promotion of branching and acinar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) for 20 days (days 15-35) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF; and iv) Induction of milk protein expression by incubating mEBs (mammospheres) for 7 days (days 35-42) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0134] In a further particularly preferred embodiment of the present invention, a method for producing a human milk-like product is provided, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), wherein step A)i) is defined as follows: i) generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days (day 2-day 0) in mTeSRTM in standard iPSC medium E8 (containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL as described in Nat Methods, 2011), and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 6 days (day 0-day 6) in MammoCultB medium supplemented with MammoCult growth supplements, hydrocortisone, heparin and BMP4 as described herein, where step A)ii) is differentiated into further sub-steps and the following steps ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I suspended in EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP) and RA for 5 days (days 6-11); iii) promoting branching and acinar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) for 15 days (days 11-26) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF; and iv) Induction of milk protein expression by incubating mEBs (mammospheres) for 5 days (days 26-31) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0135] Step iv) preferably results in differentiation into milk protein expressing cells, in particular lactocytes, and / or mammary gland-like organoids.

[0136] The standard iPSC medium E8 referred to herein (containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, as described in Chen et al., Nat Methods, 2011) is commercially available, for example as "Essential 8™ Medium" from ThermoFischer Scientific, catalog number A1517001 (see also https: / / www.thermofisher.com / order / catalog / product / A1517001# / A1517001).

[0137] mTeSR™ medium is commercially available from STEMCELL Technologies, catalog number 85850 (see also https: / / www.stemcell.com / mtesr1.html). Suchc medium is also described in "Defined, Feeder Independent medium for human hembryonic stem cell culture", Current protocols in Stem Cell Biology, Volume 2, Issue 1, September 2007.

[0138] In one embodiment, steps iii) and / or iv) as defined above for the particularly preferred embodiment preferably result in the formation / differentiation into at least breast cells, luminal cells and basal cells. In this context, the breast cells preferably express one or more, preferably all, of the markers selected from the group consisting of β-casein, milk proteins and hormone receptors. Furthermore, the luminal cells preferably express one or more, preferably all, of the markers selected from the group consisting of EpCAM, MUC1, CD49F, GATA3, CK8 and CK18. Furthermore, the basal cells preferably express one or more markers selected from the group consisting of CK14, α-smooth muscle actin and P63.

[0139] In a further embodiment, after induction of mEBs (mammospheres) in steps ii) and / or iv) as defined above for the particularly preferred embodiment, mammary-like organoids expressing one or more markers selected from the group consisting of β-casein, milk proteins and hormone receptors, luminal cells expressing one or more markers selected from the group consisting of EpCAM, MUC1, CD49F, GATA3, CK8, CK18, and basal cells expressing one or more markers selected from the group consisting of CK14, α-smooth muscle actin and P63 can be obtained.

[0140] In one embodiment of the present invention, the above method is provided for producing a human milk-like product.

[0141] In one embodiment (of step A), delivery of nutrients and biomimetic stimuli is controlled to affect cell growth, differentiation, and tissue formation. In one embodiment (of step A), such control is performed within a bioreactor.

[0142] In the methods for producing a mammalian milk-like product described herein, when BMP4 and RA are added to the culture medium, the EBs generated in the methods described herein express one or more mammary positive progenitor cell markers. In some embodiments, the one or more mammary positive progenitor cell markers are selected from EpCAM, CD49f, MUC1, and GATA3.

[0143] In some embodiments, expression of one or more mammary positive progenitor cell markers in EBs is increased compared to the expression level of the mammary positive progenitor cell marker in EBs that are not treated with BMP4.

[0144] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more of the EBs express one or more mammary positive progenitor cell markers. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EBs express two or more mammary positive progenitor cell markers. In some embodiments, the mammary positive progenitor cell markers are selected from EpCAM, CD49f, MUC1, and GATA3.

[0145] In some embodiments, at least 35% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, at least 60% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers at an intermediate differentiation stage. In some embodiments, at least 35% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers at a pre-induction stage. In some embodiments, at least 40% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers at a post-induction stage.

[0146] In some embodiments, at least 15% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, at least 20% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers at an intermediate differentiation stage. In some embodiments, at least 15% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers at a pre-induction stage. In some embodiments, at least 5% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers at a post-induction stage.

[0147] In some embodiments, at least 20% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers. In some embodiments, at least 50% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers at an intermediate differentiation stage. In some embodiments, at least 25% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers at a pre-induction stage. In some embodiments, at least 15% of the EBs express the GATA3 and EpCAM mammary positive progenitor cell markers at a post-induction stage.

[0148] In some embodiments, the intermediate differentiation stage is during day 25. The pre-induction stage is during day 35 and the post-induction stage is during day 42. In some embodiments, the intermediate differentiation stage is during day 20. The pre-induction stage is during day 26 and the post-induction stage is during day 31.

[0149] In some embodiments, the EBs express one or more milk-specific bioactive markers, hi some embodiments, the milk-specific bioactive marker is osteopontin (OPN).

[0150] In some embodiments, expression of one or more milk-specific bioactive markers in EBs is increased compared to the expression level of the non-neuronal ectodermal marker in EBs that are not treated with BMP4 and RA.

[0151] Thus, by way of example, there is provided herein a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating the iPSCs into non-neurectodermal cells by culturing the iPSCs in an appropriate culture medium (e.g., MammoCult medium) and harvesting the formed mammospheres after 10 days of BMP4 as described herein; and ii) growing such mammospheres in a suitable system (e.g., the floating mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012) for at least 10 days to generate lactocytes; wherein the expression of one or more mammary positive progenitor cell markers in the mammospheres (EBs) is increased compared to the expression level of the mammary positive progenitor cell markers in mammospheres (EBs) not treated with BMP4;

[0152] Thus, by way of example, there is provided herein a method for producing a human milk-like product, comprising generating lactocytes from human induced pluripotent stem cells (hiPSCs) under step A), said step A) comprising: i) differentiating the iPSCs into non-neuroectodermal cells by culturing the iPSCs in an appropriate culture medium (e.g., MammoCult medium) and harvesting the formed mammospheres after 6 days of BMP4 as described herein; and ii) growing such mammospheres in a suitable system containing RA (e.g., the floating mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012) for less than 10 days to generate lactocytes; Here, the expression of one or more mammary positive progenitor cell markers in the mammospheres (EBs) is increased compared to the expression levels of the mammary positive progenitor cell markers in mammospheres (EBs) that have not been treated with BMP4 and RA.

[0153] It will be appreciated that any of the methods or method steps disclosed herein may be performed in three-dimensional suspension culture rather than using a membrane matrix as a support. Thus, in some embodiments, cells are maintained in suspension culture during the entire differentiation procedure.

[0154] Step B. Expression of human breast milk-like products In one embodiment of the present invention, the method comprises expressing human milk-like products from mammary-like organoids derived from human induced pluripotent stem cells (hiPSCs), preferably prepared according to step A).The expression of human milk-like products preferably occurs upon induction of the expression of human milk-like products from such lactocytes and / or mammary-like organoids.

[0155] In one embodiment, lactating lactocytes are induced by application of a specific medium (eg, EpiCultB) supplemented with lactogenic factors (eg, prolactin, hydrocortisone, and insulin).

[0156] In particular, the human milk-like product obtained from mammary-like organoids derived from human induced pluripotent stem cells (hiPSCs), preferably prepared according to step A), contains human milk bioactive substances selected from the group including or consisting of proteins, lipids or oligosaccharides, preferably human milk oligosaccharides, etc. The inventors have succeeded in identifying, inter alia, oligosaccharides (including lactose and several HMOs), lipids (including 4 fatty acids), proteins (7 detected including casein), and miRNAs (75 detected, including 11 typically detected in HBM), in particular using the particularly preferred protocol according to steps A i) to iv) implemented above.

[0157] In one embodiment, the human milk-like product obtained from mammary-like organoids derived from human induced pluripotent stem cells (hiPSCs), preferably prepared according to step A), contains human milk bioactive substances including or selected from the group consisting of oligosaccharides, lipids, proteins, exosomes and miRNA.

[0158] In another embodiment, the human milk-like product obtained from mammary-like organoids derived from human induced pluripotent stem cells (hiPSCs), preferably prepared according to step A), is preferably selected from the group consisting of lactose, 6'SL, C-4:0 fatty acid, C-8:0 fatty acid, C-10:0 fatty acid, C-14:0 fatty acid, C-15:0 fatty acid, C-16:0 fatty acid, C-16:1n7 fatty acid, C-17:0 fatty acid, C-18:0 fatty acid, C-18:1 n9 fatty acid, C-18:1 fatty acid, C-18:2 n6 fatty acid, C-20:0 fatty acid, C-20:1 n9 fatty acid, C-18:3 The present invention comprises human milk bioactives selected from the group consisting of or including n3 fatty acids, C-22:0 fatty acids, lactoferrin, albumin, prolactin, alpha S1-casein, hemoglobin subunit beta, hemoglobin subunit alpha, α-lactalbumin, alpha-2-macroglobulin, β-casein, bile salt-activated lipase, κ-casein, lactadherin, CD14, fatty acid synthase, IgA, pIgR, serum albumin, xanthine dehydrogenase, exosomes, miR-21-5p, miR-181a-5p, miR-30d-5p, miR-30b-5p, miR-22-3p, miR-146b-3p, miR-30c-5p, miR-30a-5p, miR-30e-5p, and miR-148b-3p.

[0159] In one embodiment of the present invention, the human milk-like product obtained from the mammary gland-like organoid derived from human induced pluripotent stem cell (hiPSC) is standard human milk product.In another embodiment of the present invention, the human milk-like product obtained from the mammary gland-like organoid derived from human induced pluripotent stem cell hiPSC is non-standard human milk product.

[0160] Step C. Further processing to produce a modified human breast milk-like product In one optional embodiment of the invention, the method described herein comprises an additional step C) performed on the human milk-like product obtainable from step B) which comprises subjecting such product to further processing to provide a modified human milk-like product.

[0161] In a particular embodiment, the additional processing step C) performed on the human breast milk-like product of the present invention may be selected from the group consisting of a purification step, an isolation process, an extraction process, a fractionation step, an enrichment process, an enzymatic treatment, the addition of further components (e.g. those that cannot be expressed by human mammary organoids, such as, for example, immunoglobulins, probiotics and / or minerals) or a combination thereof.

[0162] Human Milk-Like Products "Standard" human milk-like product In one embodiment of the invention, the human breast milk-like product is a "standard" human breast milk-like product, i.e. it contains the same components as human breast milk from well-nourished mothers.

[0163] The benefits of breastfeeding are well known in the scientific literature and having access to human breast milk-like products would allow such products to be used for many equally well-known health benefits.

[0164] In such embodiments, the human breast-milk-like product can be used as a breast-milk substitute in situations where actual breast-feeding is not possible.

[0165] In such embodiments, the human breast milk-like product is intended to be used, for example, to support a longer breastfeeding experience in women who have low lactation or who have stopped lactating six months after giving birth.

[0166] Similarly, human breast milk-like products are intended to be used to enable lactation even in situations where, for example, illness impairs actual lactation from the mother.

[0167] In another embodiment, the human breast milk-like product is intended for use in situations where breast milk production does not begin naturally, such as when an infant is adopted.

[0168] In one embodiment, the human milk-like product according to the present invention is not a product of human breast milk lactation as it occurs in nature.

[0169] In one embodiment, the human breast milk-like product is for use in providing optimal nutrition to infants.

[0170] In one embodiment, the human breast milk-like product is for use in providing healthy development to an infant.

[0171] In one embodiment, the human breast milk-like product is for use in preventing infection, obesity and promoting immune development in young children.

[0172] In one embodiment, the human breast milk-like product is an unmodified human breast milk-like product.

[0173] In another embodiment, the human breast milk-like product is a modified human breast milk-like product.

[0174] In one embodiment, the human milk-like product according to the present invention comprises proteins, lipids, carbohydrates, vitamins and minerals.

[0175] In another embodiment, the human milk-like product according to the present invention comprises proteins, lipids, carbohydrates, vitamins, minerals and bioactive substances.

[0176] In one embodiment the human milk-like product according to the invention comprises proteins, lipids (including linoleic acid and alpha-linolenic acid), carbohydrates, vitamins (including vitamin A, vitamin D3, vitamin E, vitamin K, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, pantothenic acid, folic acid, vitamin C and biotin), minerals (including iron, calcium, phosphorus, magnesium, sodium, chloride, potassium, manganese, iodine, selenium, copper and zinc), choline, myo-inositol and L-carnitine.

[0177] In a further embodiment, the human milk-like product according to the invention also comprises at least one biologically active substance selected in the group consisting of proliferation / growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and / or exosomes), exosome-derived biologically active substances (e.g. miRNA) and secretory IgA.

[0178] Such human breast milk-like products may be prepared according to the methods of the present invention, for example, by including step C) of adding proliferation / growth factors, cytokines, probiotics, extracellular vesicles (e.g., milk fat globules and / or exosomes), exosome-derived bioactive substances (e.g., miRNA) and secretory IgA.

[0179] In one embodiment the human breast milk-like product contains probiotics.

[0180] Such human breast milk-like products can be prepared according to the methods of the present invention, for example, by including probiotics (e.g., B. Lactis, B. Infantis, L. Ramnhosus), which can be obtained from a number of commercially available sources.

[0181] In such embodiments, the human breast milk-like product may be used to optimize gastrointestinal function and / or promote immunity.

[0182] In one embodiment, the human breast milk-like product contains secretory IgA and probiotics.

[0183] Such a human breast milk-like product may be prepared according to the methods of the invention by including step C) of adding a combination of probiotics and secretory IgA, which may be prepared as described, for example, in WO 2009 / 156301 and WO 2009 / 156367, which are incorporated herein by reference. In such an embodiment, the human breast milk-like product may be used to prevent immunoglobulin deficiency and / or in the prevention of recurrent infections in infants and young children.

[0184] "Non-standard" human milk-like products In one embodiment of the present invention, the human milk-like product may be altered from the component ratios and concentrations naturally found in human breast milk from well-nourished mothers, referred to herein as a "non-standard milk-like product."

[0185] In one embodiment, the human milk-like product according to the present invention may be selected from the group consisting of milk fortifiers, supplements, and / or human breast milk substitutes adapted for special purposes.

[0186] Human Milk Fortifiers and Human Milk Bioactive Supplements In one embodiment, the method of the present invention provides a human breast milk-like product that can be used to fortify human breast milk obtained naturally from lactating mothers or to fortify infant formula.

[0187] In another embodiment, the method of the present invention provides a human breast milk-like product that may be used as a supplement for infants or young children in need thereof.

[0188] In such embodiments, the human breast-milk-like products may be used to provide healthy development and / or reduce the risk of developing illnesses typically associated with certain conditions in infants or young children (e.g., asthma, allergies, cognitive changes, etc.) and / or promote growth, immune development, and protection from infection.

[0189] Notably, the human origin of the ingredients (especially the bioactive ingredients) in such fortifiers or supplements, combined with the fact that they are produced by the methods of the present invention, is believed to provide such ingredients with intact or enhanced functionality.

[0190] The human breast milk-like product is preferably intended for use as a fortifier. Such a human breast milk-like product is intended for use as a fortifier and can for example be prepared according to the method of the invention by including step C) of isolation and / or enrichment of (certain) bioactive substances from the human breast milk-like product obtainable from step B). Such an isolation step may be carried out via classical fractionation, enrichment and / or purification of the unmodified human breast milk-like product obtainable from step B).

[0191] A human breast milk-like product intended for use as a supplement may comprise one or more bioactive substances selected from the group consisting of human milk oligosaccharides (e.g., 2FL, 3FL, LNT, LnNT, DiFl, 6SL and / or 3SL), lipids, growth / growth factors (e.g., epidermal growth factor (EGF), heparin-binding epidermal growth factor), cytokines (e.g., transforming growth factor-β2 (TGFβ-2), IL-1, IL-2, IL-6, IL-10, IL-18, interferon gamma (INF-γ), TNF-α), extracellular vesicles (e.g., milk fat globules and / or exosomes), exosomes containing microRNA, and antimicrobial / protective bioactive substances (e.g., IgA, lactoferrin, lysozyme, lactadherin). Such a human breast milk-like product intended for use as a supplement may be prepared according to the method of the present invention, for example, by including step C) of isolating the bioactive substances from the unmodified human breast milk-like product obtainable from step B). Such an isolation step may be carried out via classical fractionation, enrichment and / or purification of the unmodified human breast milk-like product obtainable from step B).

[0192] In one embodiment, the human breast milk-like product is a supplement or milk fortifier containing fucosylated human milk oligosaccharides, such as 2FL and / or 3FL, for use in fulfilling the profile of human breast milk from women who do not secrete fucosylated oligosaccharides due to an inactive FUT2 gene.

[0193] Such a human breast milk-like product intended for use as a fortifier or supplement may be prepared according to the method of the invention, for example by including step C) of isolating and / or enriching fucosylated oligosaccharides (e.g. 2FL and / or 3FL) from the unmodified human breast milk-like product obtainable from step B).

[0194] In such embodiments, the human breast milk-like product may be used to optimize gastrointestinal function and / or promote immunity.

[0195] Human breast milk-like products for infants with genetic disorders In one embodiment, the human breast milk-like product according to the present invention may be adapted to address the specific needs of infants born with genetic disorders.

[0196] Galactosemia In such embodiments, the human breast milk-like product can be adapted to the needs of infants suffering from galactosemia, a rare genetic disorder that affects an infant's ability to metabolize galactose.

[0197] In such embodiments, the human breast milk-like product should be depleted of lactose and / or lactose containing saccharides, hi such embodiments, the human breast milk-like product may be used to provide healthy development for infants with galactosemia.

[0198] In one embodiment, a human breast milk-like product from which lactose and / or lactose-containing saccharides have been removed may be obtained according to the method of the invention by including a step C) of enzymatic treatment (lactase treatment) or a step C) of membrane fractionation and ultrafiltration of the unmodified human breast milk-like product obtainable from step B).

[0199] In another embodiment, a human breast milk-like product depleted of lactose and / or lactose-containing saccharides can be obtained according to the method of the invention by generating hiPSCs using GMO α-lactalbumin deficient human cells in step A).

[0200] Phenylketonuria In such an embodiment, the human breast milk-like product can be adapted to the needs of infants suffering from phenylketonuria (PKU), which is caused by the absence or dysfunction of phenylalanine hydroxylase, which converts phenylalanine to tyrosine. If untreated, brain toxicity leads to severe mental retardation.

[0201] In such embodiments, the human breast milk-like product should be deprived or depleted of phenylalanine.

[0202] In such embodiments, the human breast milk-like product may be used to provide healthy development for infants affected by PKU.

[0203] In one embodiment, the human breast milk-like product is depleted in phenylalanine such that the phenylalanine content is maintained below 20 mg / kg body weight of the subject receiving such product.

[0204] In one embodiment, a phenylalanine-depleted or removed human breast milk-like product can be obtained according to the method of the invention by including a step C) of enzymatic treatment (proteolysis) or filtration of the unmodified human breast milk-like product obtainable from step B).

[0205] In one embodiment, a phenylalanine-depleted human breast milk-like product can be obtained according to the method of the invention by including a step C) of enzymatic treatment (proteolysis) or filtration of the unmodified human breast milk-like product obtainable from step B).

[0206] In another embodiment, a phenylalanine-depleted human breast milk-like product can be obtained according to the method of the present invention by providing in step B) a culture medium that provides limited or zero amounts of phenylalanine, such as a culture medium containing whey-derived glycomacropeptide (GMP).

[0207] Additional Embodiments of the Invention The present invention provides aspects according to the following numbered statements:

[0208] 1. A method for producing a population of mammary gland cells, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium containing bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) to generate embryoid bodies (EBs); ii) growing the EBs to generate a population of mammary cells.

[0209] 2. The method of statement 1, wherein the culturing step i) comprises culturing the miPSCs in a three-dimensional suspension culture system, e.g., in three-dimensional suspension conditions, in MammoCult medium and BMP4, thereby differentiating the iPSCs into non-neural ectodermal cells, optionally for at least 12 days.

[0210] 3. The method of statement 1, wherein the culturing step i) comprises culturing the miPSCs in a three-dimensional suspension culture system, e.g., in three-dimensional suspension conditions, in MammoCult medium and BMP4, thereby differentiating the iPSCs into non-neural ectodermal cells, optionally for up to 8 days.

[0211] 4. The method according to statement 1 or 2, wherein the growing step ii) comprises growing the formed EBs in a three-dimensional embedding system comprising RA, e.g. a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen I, for at least 30 days, e.g. 32 days, to generate lactocytes.

[0212] 5. The method of statement 1 or 3, wherein the growing step ii) comprises growing the formed EBs in a three-dimensional embedding system comprising RA, e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen I, for up to 25 days to generate lactocytes.

[0213] 6. Step ii) is differentiated into further sub-steps and the following steps: ii) and iii): ii) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP) and RA for 5 days; iii) The method described in statement 4, comprising promoting branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) for 20 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF.

[0214] 7. Step ii) is differentiated into further sub-steps and the following steps: ii) and iii): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I suspended in EpiCultB medium supplemented with EpiCult growth supplement, parathyroid hormone (pTHrP), and RA for 5 days; iii) The method described in statement 5, comprising incubating the embedded mEBs (mammospheres) for 15 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF to promote branching and acinar differentiation and mammary cell specification.

[0215] 8. The method according to any one of statements 1 to 7, wherein the mammary gland cells are human mammary gland cells.

[0216] 9. The method of any one of statements 1 to 8, wherein BMP4 is added to the culture medium between days 0 and 10, preferably between days 0 and 3, with day 0 being the time when the iPSCs are added to the culture medium for the first time.

[0217] 10. The method according to any one of statements 1 to 9, wherein BMP4 is added to the culture medium for 3 days.

[0218] 11. The method of any one of statements 1 to 10, wherein RA is added to the culture medium between days 10 and 15, optionally between days 6 and 11, and day 0 is the time when the iPSCs are added to the culture medium for the first time.

[0219] 12. The method according to any one of statements 1 to 11, wherein RA is added to the culture medium for 5 days.

[0220] 13. The method according to any one of statements 1 to 12, wherein BMP4 is added to the culture medium at a concentration of 5 to 20 ng / mL, preferably 5 ng / mL, 10 ng / mL or 20 ng / mL.

[0221] 14. The method according to any one of statements 1 to 13, wherein RA is added to the culture medium at a concentration of 1 μM.

[0222] 15. The method of any one of statements 1 to 14, wherein the EBs express one or more mammary positive progenitor cell markers, optionally selected from EpCAM, CD49f, MUC1 and GATA3.

[0223] 16. The method of any one of statements 1 to 15, wherein expression of one or more mammary positive progenitor cell markers in the EBs is increased compared to the expression level of the mammary positive progenitor cell markers in EBs that are not treated with BMP4 and RA.

[0224] 17. The method of any one of statements 1-16, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or more of the EBs express one or more mammary positive progenitor cell markers, optionally at least 35% of the EBs express the EpCAM and CD49f mammary positive progenitor cell markers, optionally at least 15% of the EBs express the MUC1 and EpCAM mammary positive progenitor cell markers, and / or optionally at least 20% of the EBs express the EpCAM and GATA3 mammary positive progenitor cell markers.

[0225] 18. The method of any one of statements 1-17, wherein the EBs express one or more milk-specific bioactive markers, optionally osteopontin (OPN).

[0226] 19. The method of any one of statements 1-18, wherein the EB increases expression of one or more milk-specific bioactive markers, and optionally increases expression of osteopontin (OPN).

[0227] 20. The method of any one of statements 1-19, wherein the EB increases secretion of one or more milk-specific bioactive markers, and optionally increases secretion of osteopontin (OPN).

[0228] 21. The method of any one of statements 1 to 20, wherein the mammary gland cells form lactocytic mammary-like gland organoids.

[0229] 22. Use of BMP4 and RA to improve the differentiation efficiency of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells in a differentiation protocol.

[0230] 23. A method for producing a mammalian milk-like product, comprising: A) generating mammary-like glandular organoids derived from lactocytic mammalian induced pluripotent stem cells (miPSCs); B) secreting said mammalian milk-like product from said lactocytes; The method, wherein step A) comprises culturing the miPSCs in a culture medium containing BMP4 and RA.

[0231] 24. The method according to statement 23, wherein step A) is a process of 30 to 45 days, optionally a process of less than 45 days, optionally a process of less than 44 days, optionally a process of less than 35 days, optionally a process of less than 31 days.

[0232] 25. The method according to statement 23 or 24, for producing a human milk-like product, The step A) further comprises: i) differentiating the hiPSCs into non-neural ectodermal cells by culturing the hiPSCs in a suitable culture medium comprising BMP4, e.g., MammoCult medium and BMP4, in a suitable three-dimensional culture system, e.g., in three-dimensional suspension conditions, for at least 12 days; ii) growing the formed mEBs (mammospheres) in a suitable three-dimensional embedding system containing RA, for example a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen I and RA, for at least 30 days, for example 32 days, to generate lactocytes.

[0233] 26. The method according to statement 23 or 24, for producing a human milk substitute product, comprising: The step A) further comprises: i) differentiating the hiPSCs into non-neural ectodermal cells by culturing the hiPSCs in a suitable culture medium comprising BMP4, e.g., MammoCult medium and BMP4, in a suitable three-dimensional culture system, e.g., in three-dimensional suspension conditions, for up to 8 days; ii) growing the formed mEBs (mammospheres) in an appropriate three-dimensional embedding system containing RA, for example, a mixed floating gel composed of matrix proteins such as Matrigel and / or collagen I and RA, for up to 25 days to generate lactocytes.

[0234] 27. The step A)i) is defined as follows: i) generation of embryoid bodies (EBs) from said hiPSCs by incubation for 2 days in standard iPSC medium E8 containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, or in mTeSR™ medium, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 10 days in complete MammoCult medium containing basal medium, growth supplements and supplemented with BMP4, heparin and hydrocortisone; Step A) ii) is differentiated into further sub-steps and the following steps: ii), iii) and iv): ii) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP) and RA for 5 days; iii) promotion of branching and acinar differentiation and mammary cell specification by incubation of mEBs (mammospheres) for 20 days in EpiCultB medium supplemented with EpiCult growth supplements and hydrocortisone and insulin and FGF10 and HGF; and iv) Inducing milk protein expression by incubating mEBs (mammospheres) for 7 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0235] 28. Step A) i) is defined as follows: i) generation of said embryoid bodies (EBs) from said hiPSCs by incubation for 2 days in standard iPSC medium E8 containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, or in mTeSR™ medium, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 6 days in complete MammoCult medium containing basal medium, growth supplements and supplemented with BMP4, heparin and hydrocortisone; Step A) ii) is differentiated into further sub-steps and the following steps: ii), iii) and iv): ii) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP) and RA for 5 days; iii) promoting branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) for 15 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF; and iv) Induction of milk protein expression by incubating mEBs (mammospheres) for 5 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0236] 29. Step A) i) is defined as follows: i) generation of said embryoid bodies (EBs) from said hiPSCs by incubation for 2 days in standard iPSC medium E8 containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 10 days in MammoCultB medium supplemented with MammoCult growth supplements, hydrocortisone, heparin and BMP4; Step A) ii) is differentiated into further sub-steps and the following steps: ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I suspended in EpiCultB medium supplemented with EpiCult growth supplement, parathyroid hormone (pTHrP), and RA for 5 days; iii) promotion of branching and acinar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) for 20 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF; and iv) Inducing milk protein expression by incubating mEBs (mammospheres) for 7 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0237] 30. Step A) i) is defined as follows: i) generation of said embryoid bodies (EBs) from said hiPSCs by incubation for 2 days in standard iPSC medium E8 containing DMEM / F12, magnesium L-ascorbic acid-2-phosphate, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 6 days in MammoCultB medium supplemented with MammoCult growth supplements, hydrocortisone, heparin and BMP4; Step A) ii) is differentiated into further sub-steps and the following steps: ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I suspended in EpiCultB medium supplemented with EpiCult growth supplement, parathyroid hormone (pTHrP), and RA for 5 days; iii) promotion of branching and acinar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) for 15 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10 and HGF; and iv) Inducing milk protein expression by incubating mEBs (mammospheres) for 5 days in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone and β-estradiol.

[0238] 31. A method according to any one of statements 23 to 30, wherein step A) includes a method according to any one of statements 1 to 21.

[0239] 32. The method according to statements 23, 24 or 31, wherein step A) is carried out under three-dimensional suspension culture conditions.

[0240] 33. A human milk-like product obtainable by a method according to any one of statements 23 to 32.

[0241] 34. Human milk-like products according to statement 33 for use in therapy.

[0242] 35. Use of a human milk-like product according to statement 33 as a human milk substitute, optionally as a breast milk substitute.

[0243] It should be understood that the various aspects and embodiments of the detailed description as disclosed herein are illustrative of specific ways to make and use the invention, and are not intended to limit the scope of the invention when considered in conjunction with the claims and the detailed description. It should also be understood that features from aspects and embodiments of the invention may be combined with additional features from the same or different aspects and embodiments of the invention.

[0244] As used in the detailed description and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. [Brief description of the drawings]

[0245] [Figure 1] FIG. 1 shows differentiation of human induced pluripotent stem cells (hiPSCs) according to the protocol outlined in Ying Qu and applied to one alternative method in step A) of the method of the present invention. [Diagram 2] Step A) Differentiation of human induced pluripotent stem cells (hiPSCs). [Diagram 3] FIG. 1 shows step A) of the method of the present invention showing the differentiation of human induced pluripotent stem cells (hiPSCs) according to preferred and particularly preferred embodiments. [Figure 4]Figure 2 shows that 3D organotypic cultures of hiPSCs produced according to the method are highly permissive for mammary specification. Shown are mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8 and CSN2 in the 3D differentiation (42 days) protocol. Markers indicate stages from left to right: pluripotency (Nanog), lineage (ectoderm and endoderm) (TUBB3, FOXA2), basal cell / myoepithelial markers (TP63, KR-14), luminal epithelial markers (EpCAM, KRT8), and milk protein (CSN2 (casein beta)). [Diagram 5] 2D organotypic cultures of hiPSCs produced as comparative examples are shown. mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8, and CSN2 is shown during the 2D differentiation (31 day) protocol. Markers indicate stages from left to right: pluripotency (Nanog), lineage (ectoderm and endoderm) (TUBB3, FOXA2), basal cell / myoepithelial markers (TP63, KR-14), luminal epithelial markers (EpCAM, KRT8), and milk protein (CSN2 (casein beta)). [Figure 6] Schematics of various mammary differentiation protocols. The schematics summarize the various procedures for the generation of mammary progenitor cells in 42 days (a), using bone morphogenetic protein 4 (BMP4) (b), retinoic acid (RA) (c), a combination of BMP4 and RA (d), and their combination in a shortened time course (31 days) (e). The red boxes indicate the period of addition of BMP4 and / or RA. [Figure 7]Combinatorial effect of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) on mammary differentiation of induced pluripotent stem cells (iPSCs) using a 3D organoid model. a-c show flow cytometric quantification of the number of mammary positive progenitor cells using several identified markers: EpCAM, CD49f, MUC1 and GATA3 during the differentiation time course (day 25 (for control protocol) and day 20 (for shortened time course protocol)) and pre-induction phase (day 35 (for control protocol with BMP4 and RA) and day 26 (for shortened time course protocol)), and d-f show the post-induction period (day 42 (for control protocol with BMP4 and RA) and day 31 (for shortened time course protocol)). * describes the shortened modified protocol (31 days) including the combination of BMP4 and RA. [Figure 8] 4 shows the expression of various mammary epithelial markers in iPSC-derived lactocytes in the normal, full length and shortened protocols (a-h). [Figure 9] Liquid chromatography-mass spectrometry (LC-MS / MS)-based targeted proteomic analysis of osteopontin protein secretion was detected in the medium of cultures of iPS-derived mammary cells using a control differentiation method or with bone morphogenetic protein 4 (BMP4), retinoic acid (RA), and a combination of the two factors.

[0246] Experimental section Example 1 Obtaining human milk-like products by culturing hiPSCs and differentiating them into lactocytes Ying Qu et al,Stem Cell Report vol 8,205-215 February 14 th Starting from ihPSCs, lactocytes can be cultured according to the procedure described in 2017 in order to recover the human milk-like products secreted thereby and to use them in the therapy according to the invention and / or as a breast milk substitute.

[0247] Example 2 Obtaining a human milk-like product by culturing hiPSCs and differentiating them into three-dimensional lactocytes Starting from hiPSCs according to the method of the invention, lactocytes are cultured according to steps A) and B) above and the human milk-like product secreted thereby can be recovered and used in therapy and / or as a breast milk substitute according to the invention.

[0248] Example 3 Obtaining a human milk-like product by an alternative method of culturing hiPSCs and differentiating them into lactocytes Efficient lactocyte differentiation from hiPSCs can be obtained from alternative culture conditions, including conditions 1 to 4 described below. 1. EB-derived cells are cultured as monolayers on vitronectin-coated plates in 2D culture for at least 28 days in RPMI 1640 medium containing L-glutamine, fetal bovine serum (FBS), insulin, epidermal growth factor (EGF), hydrocortisone, and Pen-Strep (penicillin / streptomycin: antibiotic-antimycotic solution). 2. Two-dimensional culture on vitronectin-coated plates of adherent aggregates of cells derived from EBs (EBPs) for at least 28 days in medium containing RPMI 1640 supplemented with L-glutamine, fetal bovine serum (FBS), insulin, epidermal growth factor (EGF), hydrocortisone, and Pen-Strep (antibiotic-antimycotic solution). 3. 3D culture in suspension in MammoCult medium for at least 10 days, then culture in specific medium (e.g., EpiCultB) in the presence of parathyroid hormone for an additional 5 days, followed by culture in a mixed floating gel (e.g., Matrigel and Collagen 1) in the presence of insulin, HGF, hydrocortisone and FGF10 for 25 days. 4. EBs are cultured in 3D in suspension in MammoCult medium (ultra-low attachment plates) for at least 10 days, then in specific medium (e.g. EpiCultB) in the presence of parathyroid hormone for an additional 5 days, followed by 25 days in the presence of insulin, HGF, hydrocortisone and FGF10.

[0249] Example 4 Two- and three-dimensional differentiation of human induced pluripotent stem cell (hiPSC) line 603-based lactocytes (a) Three-dimensional differentiation of human induced pluripotent stem cell (hiPSC) line 603-based lactocytes: For three-dimensional differentiation of lactocytes, we used human induced pluripotent stem cell (hiPSC) line 603. Human induced pluripotent stem cell (hiPSC) line 603 was purchased from Fujifilm Cellular Dynamics, Inc. (FCDI). (i) For the three-dimensional differentiation protocol (according to the present invention), single cells of hiPSCs were formed into EBs (spheroids) by incubating overnight in E8 medium containing 10 uM rock inhibitor at 37° C., 5% CO2, and rotating at 95 rpm. On day 2, the medium was replaced with E8 (day -2 to day 0). The next day, the medium was replaced with Mammo1 medium (MammoCult medium supplemented with penicillin / streptomycin and growth supplements, heparin (4 μg / mL), and hydrocortisone (0.48 μg / mL)) for 10 days (day 0-10). Medium was replaced every 2 days. (ii) After differentiation, the cells were cultured in Mammo2 medium (EpiCultB + supplements, PTHrP 100 ng / mL + penicillin / streptomycin) for 5 days. The culture medium was changed every 3 days (days 10 to 15). (iii) To induce branching of epithelial structures, acinar differentiation and mammary specification, mEBs (spheroids / mammospheres) were fed with Mammo3 medium (complete EpiCultB, hydrocortisone (1 μg / mL), insulin (10 μg / mL), FGF10 (50 ng / mL), HGF (50 ng / mL) and penicillin / streptomycin) for 20 days. The medium was changed every 3 days (days 15-35). (iv) Finally, to induce the production of milk bioactives (three-dimensional), Mammo4 medium (complete EpiCultB, 10% FBS, prolactin (10 µg / mL), hydrocortisone (1 µg / mL), insulin (10 µg / mL), progesterone, β-estradiol and penicillin / streptomycin) was used for 7 days, with the medium changed every 3 days (days 35-42). During all differentiation procedures, spheroids were maintained in suspension culture (rotating at 95 rpm). The differentiation procedure was terminated on day 42. The results are represented in Figure 4.

[0250] (b) Two-dimensional differentiation of human induced pluripotent stem cell (hiPSC) line 603-based lactocytes: For two-dimensional differentiation of lactocytes, we also used human induced pluripotent stem cell (hiPSC) line 603. Human induced pluripotent stem cell (hiPSC) line 603 was purchased from Fujifilm Cellular Dynamics, Inc. (FCDI).

[0251] In the 2D differentiation protocol (used for comparison), we used Lacto medium (RPMI 1640, 20% FBS, 1 mM glutamine, 4 μg / mL insulin, 20 ng / mL EGF, 0.5 μg / mL hydrocortisone, penicillin / streptomycin) during all differentiation steps. Cells were incubated at 37° C. and 5% CO2. Medium was changed every 2 days. The results are presented in FIG. 5.

[0252] (c) Results Quantitative RT-PCR was used to capture each stage of differentiation during lactocyte induction (Figure 4, 3D differentiation; Figure 5, 2D differentiation). In both 2D and 3D settings, NanoG expression as a marker of pluripotency decreases as cells mature and differentiate. Neuroectodermal and endodermal markers, TUBB3 (Tubulin beta 3 class III) and Forkhead box protein A2 (FOXA2), are not significantly expressed in 3D format, and elevated TUBB3 is only captured in 2D settings. This demonstrates that hiPSCs are patterned into non-neurectodermal lineages and thus enriched for mammary progenitors in 3D format. We tested the expression patterns of commonly used basal cell / myoepithelial markers such as p63 (nuclear protein with homology to p53) and cytokeratin 14 (KRT-14). Both markers are significantly detectable in both systems. Furthermore, epithelial cell adhesion molecule (EpCAM) and cytokeratin 8 (KRT8) were tracked only in the three-dimensional system, while KRT8 was only partially expressed in the two-dimensional format. As a result, the three-dimensional plates in the organ-type setting expressed breast tissue markers, luminal markers, and basal markers. Such mammary-like organoids express human breast-specific proteins, including CSN2 (casein beta), milk protein peptides, and hormone receptors. Luminal cells specifically express EpCAM, MUC1, CD49F, GATA3, CK8, and CK18, whereas basal cells specifically express CK14, α-smooth muscle actin, and p63. Finally, EpCAM and CD49F double positive cells can be detected at the early progenitor stage between D10 and D35. Interestingly, CSN2 expression is only captured at the end time point (D42) in the three-dimensional organ system, but not in the two-dimensional differentiation platform.

[0253] Analysis of the secretome of mammary-like organoids demonstrated secretion of human milk-specific bioactive substances including oligosaccharides (including lactose and several HMOs), lipids (including four fatty acids), proteins (seven detected including casein), and miRNAs (75 detected including 11 typically detected in HBM), as described below.

[0254] The supernatants of primary cells were analyzed for the presence of lactose or human milk oligosaccharides according to the procedure described in Austin and Benet, Quantitative determination of non-lactose milk oligosaccharides, Analytica Chimica Acta 2018, 1010, 86-96, with minor modifications. Samples were analyzed by UHPLC and detected lactose or human milk oligosaccharides (HMOs) were quantified against a calibration curve of a mixture of lactose and seven HMOs (2'FL, 3'FL, DFL, LNT, LNnT, 3'SL and 6'SL). The method was estimated to have a limit of 0.1 mg / L. In the supernatants of primary cells, lactose (0.22 mg / L) and 6'SL (0.32 mg / L) were detected at day 42.

[0255] The fatty acids in the medium and cell supernatants were analyzed by gas chromatography coupled with a flame ionization detector. Briefly, the supernatants obtained on day 42 are analyzed to investigate the presence of fatty acids in several lipid classes. A 7890A gas chromatograph equipped with a 7693 autosampler with a preparative station module equipped with a fused silica CP-Sil 88 capillary column (100% cyanopropylpolysiloxane, 100 m, 0.25 mm inner diameter, 0.25 mm film thickness) is used with a split injector (1:25 ratio) heated at 250 °C, and a flame ionization detector operated at 300 °C. The preparation of FAMEs (fatty acid methyl esters) is performed by direct transesterification of the samples with methanolic chloridric acid. The separation of the FAMEs is performed using capillary gas chromatography-FID (GC). The identification of the FAMEs is performed by retention time (RT) and comparison with external standards. Quantification of fatty acids is performed by calculation using methyl C11:0 as internal standard. The transesterification performance of the method is controlled using TAG C13:0 as a second internal standard. After addition of the internal standard, the solution was mixed with 2 mL of methanol, 2 mL of methanol / HCl (3N) and 1 mL of hexane. After heating at 100°C / 60 min, the sample was cooled to room temperature (approximately 15 min) and the reaction was stopped by adding 2 mL of water. After centrifugation, the organic phase was directly injected into the GC.

[0256] The fatty acid results (differences observed between medium and supernatant) from the protocol of Example 4a at day 42 are shown in Table 1.

[0257] Table 1 below lists the fatty acids expressed in the cell supernatant samples.

[0258] [Table 1]

[0259] Proteins in cell supernatants were analyzed using SDS-PAGE profiling followed by band isolation for identity confirmation by LC-MSMS. For SDS-PAGE analysis, the entire amount of prepared sample was loaded onto the gel. Human milk samples were added as controls for comparison. Selected gel regions (bands) were cut and probed for human proteins by LC-MSMS. Finally, bands were subjected to in-gel trypsin digestion and analyzed by LC-MSMS. LC-MSMS data was analyzed with Peaks Studio and matched against the UniProt database of human proteins.

[0260] Table 2 below lists the best candidates for all excised bands.

[0261] [Table 2]

[0262] Exosome isolation and miRNA profiling were performed using ExoQuick polymer nets. The ExoQuick polymer acts to precipitate exosomes by forming a network, recovering all exosomes of a certain size. Once the ExoQuick mesh is formed, the exosomes are easily pelleted by simple low-speed centrifugation. The exosomes are intact, ready for protein or RNA analysis, and bioactive for functional testing. Precipitation buffer was added to the samples at a ratio of 0.25x and then vortexed. The mixture was incubated overnight at 4°C. After incubation, the samples were centrifuged at 1500xg for 30 min. The exosome pellet was resuspended by vortexing in the initial volume with Buffer XE (QIAGEN) for QC or Lysis Buffer from HTG EdgeSeq miRNA Whole Transcriptome Assay for miRNA profiling. To evaluate the isolation of extracellular vesicles (EVs), the supernatant was first centrifuged at 3000g for 15 min to remove cell pellets and debris. Then, 100 microliters of medium was used to precipitate with ExoQuick buffer (0.25x ratio) overnight at 4°C. The EV precipitate was collected by centrifugation at 1500g for 30 min. Two precipitations were performed for each sample, the first EV precipitate was resuspended in buffer XE (QIAGEN) for possible further analysis, and the second precipitate was resuspended in 50ul HTG lysis buffer only for 10x concentration, and then subjected to miRNA profiling by HTG.

[0263] For miRNA profiling, samples were used directly in the first lysis step. Thus, the entire sample was used directly and lysed in a 1:1 ratio with plasma lysis buffer. Proteinase K (1 / 10) was then added and the sample was incubated for 3 h at 50 °C and 600 rpm on a Thermomixer. EVs were resuspended in lysis buffer and lysed in the same conditions, with an additional incubation step of 10 min at 95 °C before the lysis incubation. 26 μL of lysate was processed with 70 μL of oil on an HTG processor according to the HTG EdgeSeq miRNA Whole Transcriptome Assay V2 procedure. For indexing and amplified libraries, samples were tagged with Illumina adapters and PCR-run with OneTaq® Hot Start 2X Master Mix GC Buffer (95°C 4 min; 16 cycles: 95°C 15 s, 56°C 45 s, 68°C 45 s; 68°C 10 min; hold at 4°C) and AMPure washed (2.5 ratio) on a robotic liquid handler SciClone NGS WorkStation (Perkin Elmer). Pools were obtained using our custom pooling program on a Hamilton robot. Samples were pooled based on GX Touch Tip HS quantification. In a second run, pools were manually purified with AMPure Beads (1.8 ratio) to remove possible remaining traces of primer dimers, quantified using Qubit, and the final concentration was adjusted to 2 nM. As a final step, for MiSeq sequencing, 20 pM of the pool was loaded onto the MiSeq with 5% PhiX added and sequenced on the MiSeq for 50 base single reads using the 150V3 kit.

[0264] Briefly, 974 miRNAs were detected in cell supernatants, of which more than 75 were highly expressed miRNAs in milk samples.

[0265] Table 3 below lists the top 10 highly expressed miRNAs.

[0266] [Table 3]

[0267] Our findings provide a novel iPSC-based three-dimensional organotypic model to study the regulation and development of normal mammary cell fate and function, and breast milk bioactive substance production.

[0268] Example 5 A different method using bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) for 42 and 31 days as time-saving protocols to generate milk bioactives closely resembling those found in human breast milk was established in a three-dimensional platform using iPSC-derived mammary organoids as a biomimetic model of the human mammary gland (Figure 6).

[0269] A 42-day protocol and a shortened time-course protocol (31-day--) using 20ng / mL BMP4 and 1μM RA in single or combined formats * Figure 7 shows that 3D iPSC cells in a shortened modified differentiation protocol (31 days) including a combination of BMP4 and RA can be induced to express mammary progenitor markers such as EpCAM (CD326), CD49f, MUC1 (CD227) and GATA3, or maintain their expression profile during the differentiation time course and / or pre-induction stages (Figure 7a-c) and post-induction periods (Figure 7d-f) when compared to control conditions using flow cytometric quantification.

[0270] We evaluated the expression of various mammary epithelial markers in iPSC-derived lactocytes in the normal length and shortened protocols (Figure 8). The shortened protocol induces RELA as a specific gene to determine the lineage of mammary epithelial cells during iPSC differentiation (Figure 8a). In addition to mature luminal markers such as GATA3, specific lactocyte markers such as EpCAM, KRT8 / 18 are also induced in the shortened protocol (Figure 8b-e). In addition, ESRRA levels are induced when we switch to the 31-day protocol (Figure 8f). Similarly, shortened differentiation progression is associated with increased levels of mammary progenitor markers such as CD24 and ITGA6 (CD49f) (Figure 8g-h).

[0271] Human osteopontin peptides -GDSVVYGLR and -YPDAVATWLNPDPSQK were specifically detected in cell culture supernatants by LC-MS / MS (Figure 9).

[0272] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

Claims

1. 1. A method for producing a population of mammary gland cells, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium containing bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) to generate embryoid bodies (EBs); ii) growing the EBs to generate a population of mammary cells.

2. 2. The method of claim 1, wherein said culturing step i) comprises culturing said miPSCs in MammoCult medium and BMP4 in a three-dimensional suspension culture system, thereby differentiating said iPSCs into non-neural ectodermal cells, optionally for at least 12 days.

3. 2. The method of claim 1, wherein said culturing step i) comprises culturing said miPSCs in MammoCult medium and BMP4 in a three-dimensional suspension culture system, thereby differentiating said iPSCs into non-neural ectodermal cells, optionally for up to 8 days.

4. 2. The method of claim 1, wherein said growing step ii) comprises growing the formed EBs in a three-dimensional embedding system containing RA for at least 30 days to generate lactocytes.

5. 2. The method of claim 1, wherein the growing step ii) comprises growing the formed EBs in a three-dimensional embedding system containing RA for up to 25 days to generate lactocytes.

6. Step ii) is distinguished into further sub-steps and the following steps: ii) and iii): ii) 5-day incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult growth supplement and parathyroid hormone (pTHrP) and RA; iii) promoting branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FGF10 and HGF for 20 days.

7. Step ii) is distinguished into further sub-steps and the following steps: ii) and iii): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and collagen I suspended in EpiCult B medium supplemented with EpiCult growth supplement, parathyroid hormone (pTHrP), and RA for 5 days; iii) promoting branching and acinar differentiation and mammary cell specification by incubating the embedded mEBs (mammospheres) for 15 days in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FGF10 and HGF.

8. The method of claim 1 , wherein the mammary gland cells are human mammary gland cells.

9. 2. The method of claim 1, wherein BMP4 is added to the culture medium between days 0 and 10, day 0 being the time when the iPSCs are first added to the culture medium.

10. The method of claim 1, wherein BMP4 is added to the culture medium for 3 days.

11. 2. The method of claim 1, wherein RA is added to the culture medium between days 10 and 15, with day 0 being the time when the iPSCs are first added to the culture medium.

12. 10. The method of claim 1, wherein RA is added to the culture medium for 5 days.

13. The method of claim 1, wherein BMP4 is added to the culture medium at a concentration of 5 to 20 ng / mL.

14. 2. The method of claim 1, wherein RA is added to the culture medium at a concentration of 1 μM.

15. The method of claim 1, wherein the EBs express one or more mammary positive progenitor cell markers selected from EpCAM, CD49f, MUC1, and GATA3.

16. The method of claim 1, wherein the expression of one or more mammary gland-positive progenitor cell markers in the EBs is increased compared to the expression level of the mammary gland-positive progenitor cell markers in EBs that have not been treated with BMP4 and RA.

17. 2. The method of claim 1, wherein at least 10% of the EBs express one or more mammary positive progenitor cell markers, optionally at least 35% of the EBs express the EpCAM and CD49f mammary positive progenitor cell markers, optionally at least 15% of the EBs express the MUC1 and EpCAM mammary positive progenitor cell markers, and / or optionally at least 20% of the EBs express the EpCAM and GATA3 mammary positive progenitor cell markers.

18. 10. The method of claim 1, wherein the EBs express one or more milk-specific bioactive markers, optionally osteopontin (OPN).

19. 10. The method of claim 1, wherein the EBs have increased expression of one or more milk-specific bioactive markers, and optionally have increased expression of osteopontin (OPN).

20. 10. The method of claim 1, wherein the EBs have increased secretion of one or more milk-specific bioactive markers, and optionally have increased secretion of osteopontin (OPN).

21. 2. The method of claim 1, wherein the mammary gland cells form lactocytic mammary-like glandular organoids.

22. A culture medium comprising bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) for use in improving the efficiency of differentiation of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells in a differentiation protocol.

23. 1. A method for producing a mammalian milk-like product, comprising: A) generating lactocytic mammary-like glandular organoids derived from mammalian induced pluripotent stem cells (miPSCs); B) secreting the mammalian milk-like product from the lactocytes; The method, wherein step A) comprises culturing the miPSCs in a culture medium containing bone morphogenetic protein 4 (BMP4) and retinoic acid (RA).

24. 24. The method of claim 23, wherein step A) is a 30 to 45 day process.

25. 1. A method for producing a human milk-like product, comprising: Step A) further comprises: i) differentiating human induced pluripotent stem cells (hiPSCs) into non-neurectodermal cells by culturing the hiPSCs in a suitable three-dimensional culture system in a suitable culture medium containing BMP4 for at least 12 days; and ii) growing the formed mEBs (mammospheres) in an appropriate three-dimensional embedding system containing RA for at least 30 days to generate lactocytes.

26. 1. A method for producing a human milk replacer product, comprising: Step A) further comprises: i) differentiating human induced pluripotent stem cells (hiPSCs) into non-neural ectodermal cells by culturing the hiPSCs in a suitable three-dimensional culture system in a suitable culture medium containing BMP4 for up to 8 days; ii) growing the formed mEBs (mammospheres) in an appropriate three-dimensional embedding system containing RA for up to 25 days to generate lactocytes.

27. Step A)i) is defined as follows: i) DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO ３ generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days in standard iPSC medium E8 containing hiPSCs and transferrin, TGFβ1 or NODAL, or in mTeSR™ medium, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 10 days in complete MammoCult medium containing basal medium, growth supplements, and supplemented with BMP4, heparin, and hydrocortisone; Step A) ii) is distinguished into further sub-steps and the following steps: ii), iii) and iv): ii) 5-day incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult growth supplement and parathyroid hormone (pTHrP) and RA; iii) Promotion of branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) for 20 days in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FGF10, and HGF; and iv) inducing milk protein expression by incubating mEBs (mammospheres) for 7 days in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol.

28. Step A) i) is defined as follows: i) DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO ３ generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days in standard iPSC medium E8 containing hiPSCs and transferrin, TGFβ1 or NODAL, or in mTeSR™ medium, and generation of mEBs (mammospheres) highly enriched in non-neurectodermal cells by incubation of EBs for 6 days in complete MammoCult medium containing basal medium, growth supplements, and supplemented with BMP4, heparin, and hydrocortisone; Step A) ii) is distinguished into further sub-steps and the following steps: ii), iii) and iv): ii) 5-day incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult growth supplement and parathyroid hormone (pTHrP) and RA; iii) Promotion of branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) for 15 days in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FGF10, and HGF; and iv) inducing milk protein expression by incubating mEBs (mammospheres) for 5 days in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol.

29. Step A) i) is defined as follows: i) DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO ３ generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days in standard iPSC medium E8 containing hiPSCs and transferrin, TGFβ1, or NODAL, and generation of mEBs (mammospheres), highly enriched in non-neurectodermal cells, by incubation of EBs for 10 days in MammoCultB medium supplemented with MammoCult growth supplement, hydrocortisone, heparin, and BMP4; Step A) ii) is distinguished into further sub-steps and the following steps: ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and collagen I suspended in EpiCult B medium supplemented with EpiCult growth supplement and parathyroid hormone (pTHrP) and RA for 5 days; iii) Promotion of branching and acinar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) for 20 days in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FGF10, and HGF; and iv) inducing milk protein expression by incubating mEBs (mammospheres) for 7 days in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol.

30. Step A) i) is defined as follows: i) DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO ３ generation of embryoid bodies (EBs) from hiPSCs by incubation for 2 days in standard iPSC medium E8 containing hiPSCs and transferrin, TGFβ1, or NODAL, and generation of mEBs (mammospheres), highly enriched in non-neurectodermal cells, by incubation of EBs for 6 days in MammoCultB medium supplemented with MammoCult growth supplement, hydrocortisone, heparin, and BMP4; Step A) ii) is distinguished into further sub-steps and the following steps: ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and collagen I suspended in EpiCult B medium supplemented with EpiCult growth supplement and parathyroid hormone (pTHrP) and RA for 5 days; iii) Promotion of branching and acinar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) for 15 days in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FGF10, and HGF; and iv) inducing milk protein expression by incubating mEBs (mammospheres) for 5 days in EpiCult B medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol.

31. The method according to any one of claims 23 to 30, wherein step A) comprises a method according to any one of claims 1 to 21.

32. 24. The method of claim 23, wherein step A) is carried out in three-dimensional suspension culture conditions.

33. 32. A human milk-like product obtainable by the method of claim 31.

34. 34. The human milk-like product of claim 33 for use in therapy.

35. The human milk-like product of claim 33 for use as a human milk substitute, optionally as a breast milk substitute.

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