Method for producing a milk-like product

Abstract

A method for producing mammary cells, and a method 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 from 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 an in vitro method for producing mammalian milk-like products, for example human milk-like products, comprising: producing lactocytes from mammalian induced pluripotent stem cells (hiPSCs), for example human induced pluripotent stem cells (hiPSCs), by culture and differentiation, and / or producing mammary gland-like organoids comprising such lactocytes, and expressing mammalian milk-like products, for example human milk-like products, from such lactocytes and / or mammary gland-like organoids.The present invention also relates to mammalian milk-like products, for example 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 is becoming increasingly clear that human breast milk is the most suitable source of nutrition, at least up to the age of 6 months. Many components of human milk are not found at all, are barely found, or are less active in the cow's milk that is the basis for the manufacture of infant formulas. Such components include, for example, the protein lactoferrin, proliferation / growth factors, long-chain polyunsaturated fatty acids, or oligosaccharides. Human milk compositions are used as the gold standard for developing current infant formulas, but despite recent major developments in infant formula compositions, it is unrealistic to think that a replica of human milk can be achieved with current manufacturing processes.

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

[0004] Stem cells have been found in mammalian milk, particularly human milk, and are called human breast milk stem cells (HBsCs). hBSCs have been shown to be highly plastic and differentiate in culture into multiple cell types, and more importantly into the three lineages necessary to form the lobulo-alveolar architecture of the human mammary gland (Hassiotou F. et al. Stem Cells. 2012). However, the use of hBSCs to produce human breast milk is neither practical nor sustainable, as it requires 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 improve the method 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 produce 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, the method comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium containing bone morphogenetic protein 4 (BMP4) to generate embryoid bodies (EBs); and ii) growing the EBs to generate a population of mammary cells; Includes.

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

[0009] Further provided herein is a method for producing a mammalian milk-like product, the method comprising: A) Generating mammalian induced pluripotent stem cell (miPSC)-derived lactocytic mammary-like gland organoids; B) secreting a mammalian milk-like product from said lactocytes; Step A) comprises culturing miPSCs in a culture medium containing BMP4.

[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 for use in therapy.

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

[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" refers to secretory epithelial cells that express the CK18 cell marker and are derived from mammalian induced pluripotent stem cells (miPSCs), in particular from human induced pluripotent stem cells (hiPSCs). Human induced pluripotent stem cells (hiPSCs) as used herein are commercially available and may 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. Furthermore, suitable hiPSCs may be selected as described, for example, 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 exogenous nucleic acids and / or to include an inducible gene expression system that includes exogenous nucleic acids. 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 vary in 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 raw materials enriched with 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 in place of ingesting naturally secreted milk ("human milk substitutes"). Milk substitute products can be used as supplements ("human milk supplements") or fortifiers ("human milk fortifiers") to be ingested in combination with naturally secreted milk.

[0020] In one embodiment, the standardized human milk-like product according to the present 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 standardized human milk-like product according to the present 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 standard human milk-like product according to the invention also comprises at least one bioactive agent selected from the group consisting of proliferation / growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and / or exosomes), exosome-derived bioactive agents (e.g. miRNA), and secretory IgA. The standard human milk-like product according to the invention is not a naturally occurring human breast milk product.

[0021] In another embodiment, the human milk-like product according to the invention can be adapted to the specific needs of the infant consuming the 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 constitutional 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 offered 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 "increasing the differentiation efficiency and maturation of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells" refers to increasing the proportion of mammary progenitor cells compared to non-mammary progenitor cells generated from a starting population of miPSCs that has undergone a differentiation protocol. In the present invention, the differentiation protocol includes the use of BMP4. 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 miPSCs that has undergone an otherwise identical differentiation protocol that does not include the use of BMP4.

[0049] In the context of the present invention, the term "mammary progenitor cell" or similar terms refers to a cell that expresses at least two mammary progenitor cell 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 terms refers to a cell that does not express at least two mammary progenitor cell markers.

[0050] References herein to EpiCult Medium or EpiCultB Medium refer to serum-free culture medium containing hydrocortisone, insulin, FGF10 and HGF.

[0051] Culture media as disclosed herein refers to solids, semi-solids or liquids 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.

[0052] Methods and uses according to the present invention Manufacture of mammalian mammary cells The present invention relates to a method for producing mammary gland cells using iPSCs that have been cultured and differentiated under specific conditions.

[0053] Surprisingly, it has been demonstrated in the present invention that the addition of bone morphogenetic protein 4 (BMP4) in the methods described herein increases the proportion of non-neural ectodermal progenitor cells and decreases the proportion of neuroectodermal progenitor cells, thereby providing a more homogenous mixture of non-neural ectodermal progenitor cells.

[0054] It has also been demonstrated herein that the addition of BMP4 in the methods described herein increases the efficiency of differentiation of iPSCs into mammary progenitor cells, thus increasing the number of mammary progenitor cells produced by the methods described herein.The advantages include a higher yield of mammary cells.

[0055] Thus, the present invention provides a method for producing a population of mammary gland cells, the method comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium containing bone morphogenetic protein 4 (BMP4) to generate embryoid bodies (EBs); and ii) growing the EBs to generate a population of mammary cells; Includes.

[0056] The present invention also provides the use of BMP4 to increase the efficiency of differentiation of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells in a differentiation protocol.

[0057] In some embodiments, mammary gland cells are human mammary gland cells.In some embodiments, mammary gland cells form lactocyte-like mammary gland organoids.Said lactocyte-like mammary gland organoids are lactogenic, i.e., can produce milk.

[0058] 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 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.

[0059] In some embodiments, BMP4 is added to the culture medium for three days.

[0060] 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.

[0061] In some embodiments, BMP4 is added to the culture medium between days 0 and 3 at a concentration of 5-20 ng / mL.

[0062] EBs generated by 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.

[0063] In some embodiments, the EBs have increased levels of expression of one or more mammary positive progenitor cell markers compared to expression of said mammary positive progenitor cell markers in EBs that are not treated with BMP4.

[0064] 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.

[0065] In some embodiments, at least 50% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 5 ng / mL and at least 50% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, at least 75% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL and at least 75% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers.

[0066] In some embodiments, at least 15% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 5 ng / mL and at least 15% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers. In some embodiments, at least 20% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL and at least 20% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers.

[0067] In some embodiments, at least 20% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 5 ng / mL and at least 20% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, at least 35% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL and at least 35% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers.

[0068] In some embodiments, at least 15% of the EBs express the GATA3 mammary positive progenitor cell marker. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL, and at least 15% of the EBs express the GATA3 mammary positive progenitor cell marker.

[0069] EBs also express non-neural ectodermal markers, thereby indicating enrichment for non-neural lineages, hi some embodiments, the one or more non-neural ectodermal markers are selected from TFAP2A and TFAP2C.

[0070] In some embodiments, the EBs have at least a two-fold increase in expression of one or more non-neural ectodermal markers compared to the expression levels of said non-neural ectodermal markers in EBs that are not treated with BMP4.

[0071] In some embodiments, the EBs have at least a 3-15 fold increase in expression of one or more non-neural ectodermal markers compared to the expression levels of said non-neural ectodermal markers in EBs that are not treated with BMP4.

[0072] In some embodiments, the EBs have at least a two-fold increase in expression of the non-neural ectodermal marker TFAP2A compared to the expression level of said non-neural ectodermal marker in EBs that are not treated with BMP4, hi some embodiments, the EBs have at least a two-fold increase in expression of the non-neural ectodermal marker TFAP2C compared to the expression level of said non-neural ectodermal marker in EBs that are not treated with BMP4.

[0073] In some embodiments, the EBs have reduced expression of one or more neuroectodermal markers, hi some embodiments, the one or more neuroectodermal markers are selected from PAX6, OTX2, and SOX11.

[0074] Thus, the methods described herein provide a more homogenous cell population with respect to the types of cells present in the cell population, i.e., the cell population comprises a more homogenous population of non-neural ectodermal lineage cells.

[0075] In some embodiments, the EBs have at least 0.5-fold decreased expression of one or more neuroectodermal markers compared to the expression levels of said neuroectodermal markers in EBs not treated with BMP4, i.e., the expression levels of these markers are reduced by half, hi some embodiments, said neuroectodermal markers are selected from PAX6, OXT2, and SOX11.

[0076] In some embodiments, 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 EBs have increased expression levels of one or more milk-specific bioactivity markers compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0078] In some embodiments, the EBs have at least a two-fold increase in expression of one or more milk-specific bioactive markers compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0079] In some embodiments, the EBs have at least two-fold increased expression of OPN compared to the expression levels of the non-neural ectodermal markers in EBs not treated with BMP4. In some embodiments, BMP4 is added to the culture medium at a concentration of 5 to 20 ng / mL, and the EBs have at least two-fold increased expression of OPN compared to the expression levels of the non-neural ectodermal markers in EBs not treated with BMP4.

[0080] In some embodiments, the EBs have at least a 4-fold increase in expression of OPN compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0081] In some embodiments, the EBs have at least an 18-fold increase in expression of OPN compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0082] In some embodiments, the mammary cells undergo an increase in the expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4. In some embodiments, the mammary cells undergo at least a two-fold increase in the expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4. In some embodiments, the markers are selected from estrogen-related receptor alpha (ESRRA), keratin 14 (KRT14), and MUC1. In some embodiments, the mammary cells undergo at least a two-fold increase in the expression of ESRRA compared to mammary cells that have not been treated with BMP4. In some embodiments, the mammary cells undergo at least a two-fold increase in the expression of KRT14 compared to mammary cells that have not been treated with BMP4. In some embodiments, the mammary cells undergo at least a two-fold increase in the expression of MUC1 compared to mammary cells that have not been treated with BMP4.

[0083] The methods for producing mammary cell populations described herein may be combined with and / or used in the methods for producing mammalian milk-like products described herein, particularly as part of step A) described herein.

[0084] For example, in some embodiments of the methods for producing a mammary cell population described herein, the culturing step i) comprises culturing the miPSCs in MammoCult medium and BMP4 in a three-dimensional suspension culture system, e.g., in three-dimensional suspension conditions, thereby differentiating the iPSCs into non-neural ectodermal cells, optionally for at least 12 days.

[0085] In some embodiments, in some embodiments of the methods for producing a mammary cell population described herein, the growing step ii) comprises growing the formed EBs in a three-dimensional embedding system, for example in a mixed floating gel composed of matrix proteins such as Matrigel and / or Collagen I, for at least 30 days, for example 32 days, to generate lactocytes.

[0086] In some embodiments of the methods for producing a mammary cell population described herein, the culture step i) comprises culturing the miPSCs in MammoCult medium and BMP4 in a three-dimensional suspension culture system, e.g. in three-dimensional suspension conditions, optionally for at least 12 days, thereby differentiating the iPSCs into non-neural ectodermal cells, and the growth step ii) comprises growing the formed EBs in a three-dimensional embedding system, e.g. in 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.

[0087] Production of mammalian milk-like products The present invention also 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 the optional step C) as defined herein. Said method for producing a mammalian milk-like product as defined herein may also comprise, as part of step A), a method for producing mammalian mammary cells. In particular, BMP4 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).

[0088] 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).

[0089] Such mammary-like cells and / or organoid structures may be generated according to any of the reported methods utilizing iPSCs.

[0090] In one embodiment, such mammary gland-like cells and / or organoid structures may be generated according to the procedures described in Ying Qu et al., Stem Cell Reports, Vol. 8, 205-215, which is incorporated herein in its entirety.

[0091] More precisely, the method described in the above mentioned scientific publication (hereinafter also referred to as the "Ying Qu publication" or Ying Qu, et al. (2017)) represents a two-step protocol for generating human mammary-like cells and / or organoids from iPSCs.

[0092] Such a protocol preferably includes, as a first step (step 1), differentiation and enrichment of non-neural ectodermal 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.

[0093] In step 1, differentiation and enrichment of non-neural ectodermal 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-neural ectodermal cells.

[0094] In some embodiments, BMP4 is added to the culture medium in step 1 to increase the proportion of mammary progenitor cells as described herein.

[0095] BMP4 is added to the culture medium as described herein.

[0096] BMP4 is added to the culture conditions 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 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.

[0097] In some embodiments, BMP4 is added to the culture medium for three days.

[0098] 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.

[0099] In some embodiments, BMP4 is added to the culture medium between days 0 and 3 at a concentration of 5-20 ng / mL.

[0100] In step 2, mammary-like organoids are generated by first preparing three-dimensional cultures based on floating mixed gels (e.g., Matrigel and Collagen I) 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, insulin, 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 adding prolactin, hydrocortisone, and insulin to complete EpiCultB medium supplemented with BSA (lactocyte medium) and culturing for 5 days. The process of Ying Qu, et al. (2017) is typically completed on day 40.

[0101] 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-neuronal ectodermal cells by culturing the iPSCs in an appropriate culture medium (e.g., MammoCult medium) and harvesting mammospheres formed from the iPSCs after 10 days; and ii) growing such mammospheres in an appropriate 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; Includes.

[0102] 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 PSCs into non-neuronal ectodermal cells by culturing the PSCs in an appropriate culture medium (e.g., MammoCult medium) and BMP4 as described herein and harvesting mammospheres formed from the iPSCs after 10 days; and ii) growing such mammospheres in an appropriate 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; Includes.

[0103] 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-neuronal ectodermal cells by culturing the iPSCs in a suitable culture medium (e.g., MammoCult medium) in non-adherent conditions for mammosphere formation; and ii) growing such mammospheres in a suitable three-dimensional system (e.g., mixed floating gels composed of matrix proteins such as Matrigel and / or collagen, or suspension cultures in non-adherent plates) for at least 10 days to generate lactocytes; Includes.

[0104] 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-neuronal ectodermal cells by culturing the 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., mixed floating gels composed of matrix proteins such as Matrigel and / or collagen, or suspension cultures in non-adherent plates) for at least 10 days to generate lactocytes; Includes.

[0105] In one embodiment, mammary cell 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).

[0106] In one embodiment of the present invention, the method comprises generating mammary-like organoids under step A).

[0107] 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 a 2D monolayer of cells, 2D with attached EBs, suspension in non-adherent plates, and mixed floating gels.

[0108] In a preferred embodiment, the mixed floating gel comprises Matrigel and Collagen I.

[0109] In another preferred embodiment, the mammospheres (mEBs) in step A) are grown for at least 15 days in a suitable system (eg, the suspension mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012).

[0110] In a more preferred embodiment, the mammospheres (mEBs) in step A) are grown for 20 days in a suitable system (eg, the suspension mixed gel culture system described in Hassiotou F. et al. Stem Cells. 2012).

[0111] In one embodiment the method according to the invention provides culture conditions according to step A) [e.g. under step A)i) and / or under step A)ii)], wherein the culture conditions are adjusted to generate lactocytes derived from human induced pluripotent stem cells (hiPSCs) capable of secreting a human milk-like product.

[0112] In a 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), such step A) comprising differentiating hiPSCs into mammary cells (e.g., lactocytes) in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) for at least 42 days.

[0113] In a 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 differentiating hiPSCs into mammary cells (e.g., lactocytes) for at least 42 days in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) comprising BMP4 as described herein.

[0114] 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) for at least 12 days (day -2 to day 10); and ii) growing the formed mEBs (mammospheres) in a suitable three-dimensional embedding system (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or Collagen I) for at least 30 days, preferably 32 days, to generate lactocytes; Includes.

[0115] 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-neuronal ectodermal cells by culturing the hiPSCs in a suitable culture medium as described herein (e.g., MammoCult Medium) and BMP4 in a suitable three-dimensional culture system (e.g., three-dimensional suspension conditions) for at least 12 days (day -2 to day 10); and ii) growing the formed mEBs (mammospheres) in a suitable three-dimensional embedding system (e.g., a mixed floating gel composed of matrix proteins such as Matrigel and / or Collagen I) for at least 30 days, preferably 32 days, to generate lactocytes; Includes.

[0116] 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) comprises: 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 in mTeSR™, and production of mEBs (mammospheres) highly enriched in non-neural ectodermal 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) and hydrocortisone (typically 0.48 μg / mL); and Step A) ii) is differentiated into further sub-steps and comprises the following steps: ii), iii) and iv): ii) incubating the mEBs (mammospheres) for 5 days (days 10-15) in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP); iii) promoting branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) for 20 days (days 15-35) 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 (days 35-42) in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol; Includes.

[0117] 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) comprises: 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 in mTeSR™, and production of mEBs (mammospheres) highly enriched in non-neural ectodermal 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 comprises the following steps: ii), iii) and iv): ii) incubating the mEBs (mammospheres) for 5 days (days 10-15) in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP); iii) promoting branching and acinar differentiation and mammary cell specification by incubating mEBs (mammospheres) for 20 days (days 15-35) 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 (days 35-42) in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol; Includes.

[0118] Step iv) preferably results in differentiation into milk protein expressing cells, in particular lactocytes, and / or mammary gland-like organoids.

[0119] In a further particularly preferred 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), wherein step A) i) comprises: 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 in mTeSR™, and production of mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs for 10 days (day 0 to day 10) in MammoCultB medium supplemented with MammoCult growth supplements, hydrocortisone, and heparin, and ii) of step A) is differentiated into further sub-steps, 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 and parathyroid hormone (pTHrP) for 5 days (days 10-15); iii) Incubating the embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10, and HGF for 20 days (days 15-35) to promote branching and acinar differentiation and mammary cell specification; and iv) Inducing milk protein expression by incubating mEBs (mammospheres) for 7 days (days 35-42) in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol; Includes.

[0120] In a further particularly preferred 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), wherein step A) i) comprises: 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, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, as described in Chen et al., Nat Methods, 2011) or in mTeSR™, and production of mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs for 10 days (day 0 to day 10) in MammoCultB medium supplemented with MammoCult growth supplements, hydrocortisone, heparin, and BMP4 as described herein; and step A) ii) is differentiated into further sub-steps and includes 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 and parathyroid hormone (pTHrP) for 5 days (days 10-15); iii) Incubating the embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10, and HGF for 20 days (days 15-35) to promote branching and acinar differentiation and mammary cell specification; and iv) Inducing milk protein expression by incubating mEBs (mammospheres) for 7 days (days 35-42) in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol; Includes.

[0121] Step iv) preferably results in differentiation into milk protein expressing cells, in particular lactocytes, and / or mammary gland-like organoids.

[0122] The standard iPSC medium E8 referred to herein (comprising 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, from ThermoFischer Scientific as "Essential 8™ Medium" catalog number A1517001 (see also https: / / www.thermofisher.com / order / catalog / product / A1517001# / A1517001).

[0123] 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.

[0124] 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, breast cells preferably express one or more, preferably all, of the markers selected from the group consisting of β-casein, milk proteins and hormone receptors. Furthermore, luminal cells preferably express one or more, preferably all, of the markers selected from the group consisting of EpCAM, MUC1, CD49F, GATA3, CK8 and CK18. Furthermore, basal cells preferably express one or more markers selected from the group consisting of CK14, α-smooth muscle actin and P63.

[0125] 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 gland 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.

[0126] In one embodiment of the present invention, the above method is provided for producing a human milk-like product.

[0127] 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.

[0128] When BMP4 is added to the culture medium in the methods for producing a mammalian milk-like product described herein, 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.

[0129] In some embodiments, the EBs have increased expression of one or more mammary positive progenitor cell markers compared to the expression levels of said mammary positive progenitor cell markers in EBs that are not treated with BMP4.

[0130] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 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.

[0131] In some embodiments, at least 50% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 5 ng / mL and at least 50% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, at least 75% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL and at least 75% of the EBs express EpCAM and CD49f mammary positive progenitor cell markers.

[0132] In some embodiments, at least 15% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 5 ng / mL and at least 15% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers. In some embodiments, at least 20% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL and at least 20% of the EBs express MUC1 and CD49f mammary positive progenitor cell markers.

[0133] In some embodiments, at least 20% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 5 ng / mL and at least 20% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, at least 35% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL and at least 35% of the EBs express MUC1 and EpCAM mammary positive progenitor cell markers.

[0134] In some embodiments, at least 15% of the EBs express the GATA3 mammary positive progenitor cell marker. In some embodiments, BMP4 is added to the culture medium at a concentration of at least 20 ng / mL, and at least 15% of the EBs express the GATA3 mammary positive progenitor cell marker.

[0135] EBs also express non-neural ectodermal markers, thereby indicating enrichment for non-neural lineages, hi some embodiments, the one or more non-neural ectodermal markers are selected from TFAP2A and TFAP2C.

[0136] In some embodiments, the EBs have at least a two-fold increase in expression of one or more non-neural ectodermal markers compared to the expression levels of said non-neural ectodermal markers in EBs that are not treated with BMP4.

[0137] In some embodiments, expression of one or more non-neural ectodermal markers in EBs is increased by at least 3-15 fold compared to the expression level of said non-neural ectodermal markers in EBs that are not treated with BMP4.

[0138] In some embodiments, expression of the non-neural ectodermal marker TFAP2A in EBs is increased by at least 2-fold compared to the expression level of said non-neural ectodermal marker in EBs not treated with BMP4, hi some embodiments, expression of the non-neural ectodermal marker TFAP2C in EBs is increased by at least 2-fold compared to the expression level of said non-neural ectodermal marker in EBs not treated with BMP4.

[0139] In some embodiments, the EBs have reduced expression of one or more neuroectodermal markers, hi some embodiments, the one or more neuroectodermal markers are selected from PAX6, OTX2, and SOX11.

[0140] Thus, the methods described herein provide a more homogenous cell population with respect to the types of cells present in the cell population, i.e., the cell population comprises a more homogenous population of non-neural ectodermal lineage cells.

[0141] In some embodiments, the expression of one or more neuroectodermal markers in the EBs is reduced by at least 0.5-fold compared to the expression levels of the neuroectodermal markers in EBs that are not treated with BMP4, i.e., the expression levels of these markers are reduced by half. In some embodiments, the neuroectodermal markers are selected from PAX6, OXT2, and SOX11.

[0142] In some embodiments, EBs express one or more milk-specific bioactive markers, hi some embodiments, the milk-specific bioactive marker is osteopontin (OPN).

[0143] In some embodiments, expression of one or more milk-specific bioactive markers in EBs is increased compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0144] In some embodiments, expression of one or more milk-specific bioactive markers in EBs is increased by at least two-fold compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0145] In some embodiments, the expression of OPN in the EBs is increased by at least 2-fold compared to the expression levels of the non-neural ectodermal markers in EBs not treated with BMP4. In some embodiments, BMP4 is added to the culture medium at a concentration of 5 to 20 ng / mL, and the expression of OPN in the EBs is increased by at least 2-fold compared to the expression levels of the non-neural ectodermal markers in EBs not treated with BMP4.

[0146] In some embodiments, expression of OPN in EBs is increased by at least 4-fold compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0147] In some embodiments, expression of OPN in EBs is increased by at least 18-fold compared to the expression levels of the non-neural ectodermal markers in EBs that are not treated with BMP4.

[0148] In some embodiments, the mammary cells undergo an increase in expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4. In some embodiments, the mammary cells undergo at least a two-fold increase in expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4. In some embodiments, the markers are selected from estrogen-related receptor alpha (ESRRA), keratin 14 (KRT14), and MUC1. In some embodiments, the mammary cells undergo at least a two-fold increase in expression of ESRRA compared to mammary cells that have not been treated with BMP4. In some embodiments, the mammary cells undergo at least a two-fold increase in expression of KRT14 compared to mammary cells that have not been treated with BMP4. In some embodiments, the mammary cells undergo at least a two-fold increase in expression of MUC1 compared to mammary cells that have not been treated with BMP4.

[0149] 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), such step A) comprising: i) differentiating the iPSCs into non-neuronal ectodermal cells by culturing the iPSCs in a suitable culture medium as described herein (e.g., MammoCult medium) and BMP4, and harvesting mammospheres formed from the iPSCs after 10 days; and ii) growing such mammospheres in an appropriate 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; and wherein 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.

[0150] 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.

[0151] 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.

[0152] In one embodiment, lactating lactocytes are induced by application of a specific medium (eg, EpiCultB) supplemented with lactogenic factors (eg, prolactin, hydrocortisone, and insulin).

[0153] 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. Using the particularly preferred protocol according to steps Ai)-iv) implemented above, the inventors have been able to identify, 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).

[0154] 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.

[0155] 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 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] In one embodiment, the human breast milk-like product is for use in providing optimal nutrition to infants.

[0166] In one embodiment, the human breast milk-like product is for use in providing healthy development to an infant.

[0167] In one embodiment, the human breast milk-like product is for use in preventing infection, obesity and promoting immune development in young children.

[0168] In one embodiment, the human breast milk-like product is an unmodified human breast milk-like product.

[0169] In another embodiment, the human breast milk-like product is a modified human breast milk-like product.

[0170] In one embodiment, the human milk-like product according to the present invention comprises proteins, lipids, carbohydrates, vitamins and minerals.

[0171] In another embodiment, the human milk-like product according to the present invention comprises proteins, lipids, carbohydrates, vitamins, minerals and bioactive substances.

[0172] 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.

[0173] In a further embodiment, the human milk-like product according to the present invention also comprises at least one biologically active substance selected in the group consisting of growth / 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.

[0174] 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.

[0175] In one embodiment the human breast milk-like product contains probiotics.

[0176] 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.

[0177] In such embodiments, the human breast milk-like product may be used to optimize gastrointestinal function and / or promote immunity.

[0178] In one embodiment, the human breast milk-like product contains secretory IgA and probiotics.

[0179] Such a human breast milk-like product may be prepared according to the methods of the invention by including step C) of adding a combination of probiotics and secretory IgA, which may be prepared as described, for example, in WO 2009 / 156301 and WO 2009 / 156367, which are incorporated herein by reference. In such an embodiment, the human breast milk-like product may be used to prevent immunoglobulin deficiencies and / or in the prevention of recurrent infections in infants and young children.

[0180] "Non-standard" human milk-like products In one embodiment of the present invention, the human milk-like product may have ingredient ratios and concentrations that are altered from those naturally found in human breast milk from well-nourished mothers, such products being referred to herein as "non-standard milk-like products."

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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).

[0187] 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 obtained 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).

[0188] 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.

[0189] 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).

[0190] In such embodiments, the human breast milk-like product may be used to optimize gastrointestinal function and / or promote immunity.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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).

[0195] In another embodiment, a human breast milk-like product depleted of lactose and / or lactose-containing saccharides can be obtained by generating hiPSCs using GMO α-lactalbumin deficient human cells under step A) according to the method of the invention.

[0196] 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.

[0197] In such embodiments, the human breast milk-like product should be deprived or depleted of phenylalanine.

[0198] In such embodiments, the human breast milk-like product may be used to provide healthy development for infants affected by PKU.

[0199] 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 consuming such product.

[0200] 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).

[0201] 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).

[0202] 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).

[0203] Additional Embodiments of the Invention The following is provided:

[0204] S1. 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) to generate embryoid bodies (EBs); and ii) growing the EBs to generate a population of mammary cells; A method comprising:

[0205] S2. The method of statement 1, wherein the culturing step i) comprises culturing the miPSCs in MammoCult medium and BMP4 in a three-dimensional suspension culture system, e.g. in three-dimensional suspension conditions, thereby differentiating the iPSCs into non-neural ectodermal cells, optionally for at least 12 days.

[0206] S3. The method according to statement 1 or 2, wherein the growing step ii) comprises growing the formed EBs in a three-dimensional embedding system, for example in a mixed floating gel composed of matrix proteins such as Matrigel and / or Collagen I, for at least 30 days, for example 32 days, to generate lactocytes.

[0207] S4. The method of any one of statements 1 to 3, wherein the mammary gland cells are human mammary gland cells.

[0208] S5. The method according to any one of statements 1 to 4, wherein BMP4 is added to the culture medium between days 0 and 10, preferably between days 0 and 3, day 0 being the time when the iPSCs are added to the culture medium for the first time.

[0209] S6. The method according to any one of statements 1 to 5, wherein BMP4 is added to the culture medium for 3 days.

[0210] S7. The method according to any one of statements 1 to 6, 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.

[0211] S8. The method of any one of statements 1-7, wherein the EBs express one or more mammary positive progenitor cell markers, optionally selected from EpCAM, CD49f, MUC1, and GATA3.

[0212] S9. The method of any one of statements 1 to 8, wherein the expression of one or more mammary positive progenitor cell markers is increased in the EBs compared to the expression level of the mammary positive progenitor cell markers in EBs not treated with BMP4.

[0213] S10. The method of any one of statements 1-9, 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 50% of the EBs express the EpCAM and CD49f mammary positive progenitor cell markers, optionally at least 15% of the EBs express the MUC1 and CD49f mammary positive progenitor cell markers, optionally at least 20% of the EBs express the MUC1 and EpCAM mammary positive progenitor cell markers, and / or optionally at least 15% of the EBs express the GATA3 mammary positive progenitor cell marker.

[0214] S11. The method of any one of statements 1-10, wherein the EBs express one or more non-neural ectodermal markers, optionally selected from TFAP2A and TFAP2C.

[0215] S12. The method of any one of statements 1-11, wherein expression of one or more non-neural ectodermal markers in said EBs is increased by at least 2-fold, and optionally by at least 3-15-fold, compared to the expression level of said non-neural ectodermal markers in EBs not treated with BMP4.

[0216] S13. The method of any one of statements 1-12, wherein expression of one or more neuroectodermal markers in the EBs is reduced by at least 0.5 fold compared to the expression level of the neuroectodermal marker in EBs not treated with BMP4, and optionally the neuroectodermal marker is selected from PAX6, OXT2, and SOX11.

[0217] S14. The method of any one of statements 1-13, wherein the EBs express one or more milk-specific bioactive markers, optionally osteopontin (OPN).

[0218] S15. The method of any one of statements 1-14, wherein the expression of OPN in the EBs is increased by at least 2-fold, and optionally by at least 4-18-fold, compared to the expression level of OPN in EBs not treated with BMP4.

[0219] S16. The method of any one of statements 1 to 15, wherein the mammary gland cells form lactocytic mammary-like gland organoids.

[0220] S17. The method of any one of statements 1-16, wherein the mammary cells undergo increased expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4, and optionally, the mammary cells undergo at least a two-fold increase in expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4, and optionally, the marker is selected from estrogen-related receptor alpha (ESRRA), keratin 14 (KRT14), and MUC1.

[0221] S18. Use of BMP4 to increase the differentiation efficiency of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells in a differentiation protocol.

[0222] S19. A method for producing a mammalian milk-like product, comprising: A) generating lactocytic mammary-like gland organoids from mammalian induced pluripotent stem cells (miPSCs); and B) secreting said mammalian milk-like product from said lactocytes; wherein step A) comprises culturing the miPSCs in a culture medium containing BMP4.

[0223] S20. A method for producing a human milk-like product, comprising: Step A) i) differentiating the hiPSCs into non-neuronal ectodermal cells by culturing the hiPSCs in a suitable culture medium comprising BMP4, e.g., in a medium comprising MammoCult medium and BMP4, in a suitable three-dimensional culture system, e.g., in three-dimensional suspension conditions, for at least 12 days; and ii) growing the formed mEBs (mammospheres) in a suitable three-dimensional embedding system, for example in a mixed floating gel composed of matrix proteins such as Matrigel and / or Collagen I, for at least 30 days, for example 32 days, to generate lactocytes; The method according to claim 19, further comprising:

[0224] S21. Step A)i) is as follows: i) generation of embryoid bodies (EBs) from 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 medium mTeSR™, and production of mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of the EBs for 10 days in complete MammoCult medium containing the basal medium, growth supplements, and supplemented with BMP4, heparin, and hydrocortisone; and Step A) ii) is differentiated into further sub-steps and comprises the following steps: ii), iii) and iv): ii) incubating the mEBs (mammospheres) for 5 days in complete EpiCultB medium supplemented with EpiCult growth supplements and parathyroid hormone (pTHrP); iii) Incubating the mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10, and HGF for 20 days to promote branching and acinar differentiation and mammary cell specification; and iv) inducing milk protein expression by incubating the mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol for 7 days; The method according to statement 20, comprising:

[0225] S22. Step A)i) is as follows: i) generation of embryoid bodies (EBs) from hiPSCs by incubating them for 2 days in standard iPSC medium E8 containing DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFβ1 or NODAL, and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubating the EBs for 10 days in MammoCultB medium supplemented with MammoCult growth supplements, hydrocortisone, heparin and BMP4, and wherein ii) of step A) is differentiated into further sub-steps and includes 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) for 5 days; iii) Incubating the embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FGF10, and HGF for 20 days to promote branching and acinar differentiation and mammary cell specification; and iv) inducing milk protein expression by incubating the mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplements, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol for 7 days; The method according to statement 20, comprising:

[0226] S23. A method according to any one of statements 19 to 22, wherein step A) includes a method according to any one of statements 1 to 17.

[0227] S24. The method according to statement 19 or 23, wherein step A) is carried out under three-dimensional suspension culture conditions.

[0228] S25. A human milk-like product obtainable according to the method described in any one of statements 19 to 24.

[0229] S26. A human milk-like product as described in statement 25 for use in therapy.

[0230] S27. Use of a human milk-like product according to statement 25 as a human milk substitute, optionally as a breast milk substitute.

[0231] 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.

[0232] 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]

[0233] [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 the differentiation of human induced pluripotent stem cells (hiPSCs) according to preferred and particularly preferred embodiments for step A) of the method of the present invention. [Figure 4]Figure 2 shows that 3D organotypic cultures of hiPSCs produced according to the method are highly permissive for mammary specification. Shown is mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8 and CSN2 during 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 6-1] Figure 1 shows the effect of bone morphogenetic protein 4 (BMP4) on non-neural ectodermal lineage differentiation of induced pluripotent stem cells (iPSCs) using a 3D organoid setup. a. Scheme outlining the procedure for mammary progenitor generation and BMP4 treatment during days 0-3. b. Microscopic observation of morphological changes after exposure to different concentrations of BMP4. c-f. Flow cytometric quantification of the number of mammary positive progenitor cells using various markers identified: EpCAM, CD49f, MUC1, and GATA3. g-h. RNA expression levels of human TFAP2A and TFAP2C as major non-neural ectodermal markers. i-k. Human PAX6, OTX2, and SOX11 as neuroectodermal markers. l. Human KRT18 as luminal marker. m. Human OPN as secreted phosphorylated lactate glycoprotein. [Figure 6-2]Figure 1 shows the effect of bone morphogenetic protein 4 (BMP4) on non-neural ectodermal lineage differentiation of induced pluripotent stem cells (iPSCs) using a 3D organoid setup. a. Scheme outlining the procedure for mammary progenitor generation and BMP4 treatment during days 0-3. b. Microscopic observation of morphological changes after exposure to different concentrations of BMP4. c-f. Flow cytometric quantification of the number of mammary positive progenitor cells using various markers identified: EpCAM, CD49f, MUC1, and GATA3. g-h. RNA expression levels of human TFAP2A and TFAP2C as major non-neural ectodermal markers. i-k. Human PAX6, OTX2, and SOX11 as neuroectodermal markers. l. Human KRT18 as luminal marker. m. Human OPN as secreted phosphorylated lactate glycoprotein. [Figure 7] The effect of BMP4-treated samples is shown. a. Compared to control samples, they showed higher expression of the dual mammary marker EpCAM+ / CD49f+ and the dual mature luminal lactocytic marker MUC1+ / EpCAM+. b. The effect of bone morphogenetic protein 4 (BMP4) on the RNA expression levels of estrogen-related receptor alpha (ESRRA), keratin 14 (KRT14), and MUC1 during days 30-35 of mammary three-dimensional differentiation.

[0234] experiment Example 1 Obtaining human milk-like products by culturing hiPSCs and differentiating them into lactocytes Ying Qu et al,Stem Cell Report vol 8,205-215 February 14 th Starting from ihPSCs, lactocytes can be cultured according to the procedure described in 2017, and the human milk-like products secreted thereby can be recovered and used in the therapy according to the invention and / or as breast milk substitute.

[0235] 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.

[0236] 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 were cultured as monolayers in two dimensions on vitronectin-coated plates and supplemented with L-glutamine, fetal bovine serum (FBS), insulin, epidermal growth factor (EGF), hydrocortisone, and Pen-Strep (Penicillin / Streptomycin: antibiotic-antimycotic solution).

[0237] 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.

[0238] 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).

[0239] (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.

[0240] (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).

[0241] (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).

[0242] (iv) Finally, to induce milk bioactive production (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.

[0243] (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).

[0244] In the 2D differentiation protocol (used for comparison), we used Lacto medium (RPMI 1640, 20% FBS, 1 mM glutamine, 4 μg / ml insulin, 20 ng / ml EGF, 0.5 μg / ml hydrocortisone, penicillin / streptomycin) during all differentiation steps. Cells were incubated at 37° C., 5% CO2. Medium was changed every 2 days. The results are presented in FIG. 5.

[0245] (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 towards non-neurectodermal lineages and thus enriched for mammary progenitors in 3D format. We tested the expression patterns of commonly used basal cell / myoepithelial markers such as p63 (nuclear protein with homology to p53) and cytokeratin 14 (KRT-14). Both markers are significantly detectable in both systems. Furthermore, epithelial cell adhesion molecule (EpCAM) and cytokeratin 8 (KRT8) were tracked only in the three-dimensional system, while KRT8 was only partially expressed in the two-dimensional format. As a result, the three-dimensional plates in the organ-type setting expressed both breast tissue markers, luminal markers, and basal markers. Such mammary-like organoids express human breast-specific proteins, including CSN2 (casein beta), milk protein peptides, and hormone receptors. Luminal cells specifically express EpCAM, MUC1, CD49F, GATA3, CK8, and CK18, whereas basal cells specifically express CK14, α-smooth muscle actin, and P63. Finally, EpCAM and CD49F double positive cells can be detected at the early progenitor stage between D10 and D35. Interestingly, CSN2 expression is only captured at the end time point (D42) in the three-dimensional organ system, but not in the two-dimensional differentiation platform.

[0246] 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.

[0247] 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.

[0248] The fatty acids in the medium and cell supernatants were analyzed by gas chromatography coupled with a flame ionization detector. Briefly, the supernatants obtained on day 42 are analyzed to investigate the presence of fatty acids in several lipid classes. A 7890A gas chromatograph equipped with a 7693 autosampler with a preparative station module equipped with a fused silica CP-Sil 88 capillary column (100% cyanopropylpolysiloxane, 100 m, 0.25 mm inner diameter, 0.25 mm film thickness) is used with a split injector (1:25 ratio) heated at 250 °C, and a flame ionization detector operated at 300 °C. The preparation of FAMEs (fatty acid methyl esters) is carried out by direct transesterification of the samples with methanolic chloridric acid. The separation of the FAMEs is carried out using capillary gas chromatography-FID (GC). The identification of the FAMEs is carried out by retention time (RT) and comparison with external standards. Quantification of fatty acids is performed by calculation using methyl C11:0 as internal standard. The transesterification performance of the method is controlled using TAG C13:0 as a second internal standard. After addition of the internal standard, the solution was mixed with 2 mL of methanol, 2 mL of methanol / HCl (3N) and 1 mL of hexane. After heating at 100°C / 60 min, the sample was cooled to room temperature (approximately 15 min) and the reaction was stopped by adding 2 mL of water. After centrifugation, the organic phase was directly injected into the GC.

[0249] The fatty acid results (differences observed between medium and supernatant) from the protocol of Example 4a at day 42 are shown in Table 1.

[0250] Table 1 below lists the fatty acids expressed in the cell supernatant samples.

[0251] [Table 1]

[0252] Proteins in cell supernatants were analyzed using SDS-PAGE profiling followed by band isolation for identity confirmation by LC-MSMS. For SDS-PAGE analysis, the entire amount of prepared sample was loaded onto the gel. Human milk sample was added as a control for comparison. Selected gel regions (bands) were cut and probed for human proteins by LC-MSMS. Finally, the bands were subjected to in-gel trypsin digestion and analyzed by LC-MSMS. The LC-MSMS data was analyzed in Peaks Studio and matched against the UniProt database of human proteins.

[0253] Table 2 below lists the best candidates for all excised bands.

[0254] [Table 2]

[0255] 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.

[0256] 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 10 min incubation step 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.

[0257] Briefly, 974 miRNAs were detected in cell supernatants, of which more than 75 were highly expressed miRNAs in milk samples.

[0258] Table 3 below lists the top 10 highly expressed miRNAs.

[0259] [Table 3]

[0260] 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.

[0261] Example 5 Effect of bone morphogenetic protein 4 (BMP4) on non-neuronal ectodermal lineage differentiation of induced pluripotent stem cells (iPSCs) using a three-dimensional organoid setting Methods: Human induced pluripotent stem cell (hiPSC) line 603 was purchased from Fujifilm Cellular Dynamics, Inc (FCDI) and used for three-dimensional differentiation of mammary progenitor cells. Briefly, 5.5 million dissociated iPSCs were seeded into 6-well plates (ultra-low attachment) containing 4.5 mL of aggregation medium using a planar shaker platform (set at 95 RPM). Different concentrations of BMP4 (314-BP-050, Bio-Techne AG) were added to the culture medium between days 0 and 3.

[0262] Results: iPSC cells were tested with various concentrations of BMP4 to induce non-neural ectodermal differentiation (Figure 6a,b). BMP4 at 20 ng / mL significantly induced the expression of mammary progenitor markers such as EpCAM (CD326), CD49f, MUC1 (CD227), and GATA3 using flow cytometry quantification (Figure 6c-f). Expression of human TFAP2A and TFAP2C (AP2 gamma) as major non-neural ectodermal markers was increased at day 10 of differentiation using Nanostring analysis compared to control samples (Figure 6g-h). Furthermore, expression levels of human specific neuroectodermal markers such as paired box gene 6 (PAX6), orthodenticle homeobox 2 (OTX2), and SRY box transcription factor 11 (SOX11) were decreased during differentiation (Figure 6i-k). Finally, BMP4 can induce the expression of human luminal specific markers cytokeratin 18 (KRT18) and secreted phosphorylated lactate glycoprotein osteopontin (OPN) (Figure 6 l-m).

[0263] These results demonstrate that BMP4 induces commitment of iPSCs towards a non-neural ectodermal lineage and, surprisingly, significantly increases mammary progenitor specification.

[0264] Example 6 BMP4-treated samples showed higher expression of the dual mammary markers EpCAM+ / CD49f+ and the dual mature luminal lactocytic markers MUC1+ / EpCAM+ compared to control samples (Figure 7a).

[0265] Furthermore, we evaluated the effect of BMP4 on the final stage of 3D mammary differentiation of iPSC cells using expression profiles of estrogen-related receptor alpha (ESRRA), keratin 14 (KRT14), and MUC1. Surprisingly, BMP4 increased the RNA expression levels of all genes at days 30, 33, and 35 compared to control samples (Figure 7b).

[0266] 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 mammary cell population, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium containing bone morphogenetic protein 4 (BMP4) to generate embryoid bodies (EBs); and ii) growing the EBs to generate a mammary cell population; A method comprising:

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-neuronal ectodermal cells, optionally for at least 12 days.

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

4. The method described in claim 3, wherein the three-dimensional embedding system is a mixed floating gel composed of matrix proteins.

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

6. 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.

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

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

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

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

11. 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 50% of the EBs express the EpCAM and CD49f mammary positive progenitor cell markers, optionally at least 15% of the EBs express the MUC1 and CD49f mammary positive progenitor cell markers, optionally at least 20% of the EBs express the MUC1 and EpCAM mammary positive progenitor cell markers, and / or optionally at least 15% of the EBs express the GATA3 mammary positive progenitor cell marker.

12. The method of claim 1, wherein the EBs express one or more non-neural ectodermal markers, optionally selected from TFAP2A and TFAP2C.

13. The method of claim 1, wherein the expression of one or more non-neural ectodermal markers in the EBs is increased by at least two-fold compared to the expression level of the non-neural ectodermal markers in EBs that are not treated with BMP4.

14. 2. The method of claim 1, wherein the expression of one or more neuroectodermal markers in the EBs is reduced by at least 0.5-fold compared to the expression level of the neuroectodermal markers in EBs not treated with BMP4, and optionally the neuroectodermal markers are selected from PAX6, OXT2, and SOX11.

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

16. The method of claim 1, wherein the expression of OPN in the EBs is increased by at least two-fold compared to the expression level of OPN in EBs not treated with BMP4.

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

18. 2. The method of claim 1, wherein the mammary cells undergo an increase in expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4, and optionally, the mammary cells undergo at least a two-fold increase in expression of a milk-specific bioactive marker compared to mammary cells that have not been treated with BMP4, and optionally, the marker is selected from estrogen-related receptor alpha (ESRRA), keratin 14 (KRT14), and MUC1.

19. A culture medium comprising bone morphogenetic protein 4 (BMP4) for use in increasing the differentiation efficiency of mammalian induced pluripotent stem cells (miPSCs) into mammary progenitor cells in a differentiation protocol.

20. 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 said mammalian milk-like product from said lactocytes; wherein step A) comprises culturing the miPSCs in a culture medium containing bone morphogenetic protein 4 (BMP4).

21. 1. A method for producing a human milk-like product, comprising: Step A) i) differentiating human induced pluripotent stem cells (hiPSCs) into non-neuronal ectodermal 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 a suitable three-dimensional embedding system for at least 30 days to generate lactocytes; 21. The method of claim 20, further comprising:

22. The method of claim 21, wherein the three-dimensional embedding system is a mixed floating gel composed of matrix proteins.

23. Step A) i) comprises: i) DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO ３ and generating embryoid bodies (EBs) from hiPSCs by incubation for 2 days in standard iPSC medium E8 containing hiPSCs and transferrin, TGFβ1 or NODAL, or in medium mTeSR™, and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubating the EBs for 10 days in complete MammoCult medium containing basal medium, growth supplements, and supplemented with BMP4, heparin, and hydrocortisone; and Step A) ii) is distinguished into further sub-steps and comprises the following steps: ii), iii) and iv): ii) incubating the mEBs (mammospheres) for 5 days in complete EpiCultB medium supplemented with EpiCult growth supplement and parathyroid hormone (pTHrP); iii) promoting 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 the mEBs (mammospheres) for 7 days in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol; 22. The method of claim 21, comprising:

24. Step A) i) comprises: i) DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO ３ and the generation of embryoid bodies (EBs) from hiPSCs by incubating them for 2 days in standard iPSC medium E8 containing MammoCult growth supplement, transferrin, TGFβ1 or NODAL, and the production of mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubating the EBs for 10 days in MammoCultB medium supplemented with MammoCult growth supplement, hydrocortisone, heparin, and BMP4, and wherein step A)ii) is distinguished into further sub-steps and includes 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) for 5 days; iii) Incubating the embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FGF10, and HGF for 20 days to promote branching and acinar differentiation and mammary cell specification; and iv) incubating the mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult growth supplement, hydrocortisone, insulin, FBS, prolactin, progesterone, and β-estradiol for 7 days to induce milk protein expression; 22. The method of claim 21, comprising:

25. The method according to any one of claims 20 to 24, wherein step A) comprises a method according to any one of claims 1 to 18.

26. 21. The method of claim 20, wherein step A) is carried out in three-dimensional suspension culture conditions.

27. 26. A human milk-like product obtainable according to the method of claim 25.

28. 28. The human milk-like product of claim 27 for use in therapy.

29. The human milk-like product of claim 27 for use as a human milk substitute, optionally as a breast milk substitute.

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