Exosomes from milk obtained by ex vivo culture of human lactocytes, and uses thereof

Abstract

The invention provides isolated human breast milk exosomes, and compositions thereof. The present invention also provides in vitro methods for producing said exosomes comprising generating lactocytes derived from human induced pluripotent stem cells (hiPSC), expressing the human milk like product from lactocytes, and purifying the exosomes from the human milk like product. The invention also provides therapeutic uses of the isolated human breast milk exosomes, and compositions thereof.

Description

[0001] Exosomes and uses thereof

[0002] Field

[0003] The present invention concerns isolated human breast milk exosomes produced by in vitro methods, and compositions comprising exosomes thereof useful for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning.

[0004] Background

[0005] Mammalian and especially human milk is a complex fluid with a multitude of components, each of which may contribute substantially to infant and perhaps maternal health. It is becoming increasingly clear that human breastmilk is the most appropriate source of nutrition at least up to the age of 6 months. Many components of human milk are simply not found or poorly found or less active in cow's milk upon which infant formula manufacture is based. This includes for instance protein lactoferrin, growth factors, long chain polyunsaturated fatty acids or oligosaccharides. Human milk composition has been used as a gold standard to develop current infant formula. However, despite recent major development in infant formula composition, it is illusory to think that human milk replication or replication of components of said human milk will be achieved with current manufacturing processes.

[0006] Today the only source of human milk is human donors (breastfeeding mothers). Donations are reported for noncommercial use (human milk biobank) and commercial use. However, this is limited and has strong regulatory, safety and sometime ethical or religious constraints.

[0007] Milk exosomes from human milk have been suggested to potentially affect development of the brain, as well as having other health benefits. It would be hugely beneficial to be able to produce exosomes by in vitro methods, where the exosomes are comparable in quality to milk exosomes produced in vivo and that can promote and / or support aspects of brain development.

[0008] Summary

[0009] The present invention solves the above-mentioned technical problem.

[0010] The present invention provides an exosome, a population of exosomes, a composition comprising exosomes or an exosome product, as described anywhere herein, that may be used for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning. This could be in a healthy subject (non-therapeutic) or in a subject with suboptimal brain development (therapeutic), such as infants who are born pre-term, are small for gestational age, are low-birth weight (LBW) or who experienced intra-uterine growth retardation (IUGR) or who suffered from growth stunting because of malnutrition, such as suboptimal intrauterine nutrition and / or disease.

[0011] Also provided herein is a method of promoting, supporting or optimising brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential, and / or cognitive functioning comprising administering to a subject in need thereof an exosome, a population of exosomes, a composition comprising exosomes or an exosome product, as described anywhere herein, to said subject. Also provided herein is a method of treating a disease or disorder selected from a demyelinating disease, a neurodevelopmental disorder, a neurocognitive disorder, or a neuropsychiatric disorder, the method comprising administering to a subject in need thereof an exosome, a population of exosomes, a composition comprising exosomes or an exosome product, as described anywhere herein, to said subject.

[0012] Provided for uses or methods described herein is an isolated human breast milk exosome, wherein the exosome does not comprise one or more proteins selected from Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1).

[0013] Also provided for uses or methods described herein is an isolated human breast milk exosome, wherein the exosome does not comprise one or more proteins selected from C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0014] Also provided for uses or methods described herein is an isolated human breast milk exosome, wherein Hypoxia up- regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome. Also provided herein is an isolated human breast milk exosome, wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome under the same conditions in which Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are detected by liquid chromatography - mass spectrometry in an exosome isolated from human breast milk.

[0015] Also provided for uses or methods described herein is an isolated human breast milk exosome, wherein C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2- oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are not detected by liquid chromatography - mass spectrometry in the exosome. Also provided herein is an isolated human breast milk exosome, wherein C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1, cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are not detected by liquid chromatography - mass spectrometry in the exosome under the same conditions in which C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are detected in an exosome isolated from human breast milk.

[0016] Also provided for uses or methods described herein is a population of exosomes, wherein the population comprises exosomes as described anywhere herein.

[0017] Also provided for uses or methods described herein is a composition comprising exosomes or a population of exosomes as described anywhere herein.

[0018] Also provided for uses or methods described herein is a human breast milk exosome product which is obtainable according to an in vitro method, the method comprising:

[0019] A) Generating lactocyte mammary-like gland organoids derived from human induced pluripotent stem cells (hiPSC) B) Secreting a human milk like product from said lactocytes, and

[0020] C) Purifying the exosomes from the human milk like product to remove impurities, optionally by chromatography or filtration or ultracentrifugation, in order to isolate the exosomes, wherein step A) comprises culturing the hiPSCs in a culture medium comprising BMP4 and / or RA.

[0021] Further, provided herein is a human milk fortifier comprising an exosome, a population of exosomes, a composition comprising exosomes, or an exosome product, as described anywhere herein, for the uses or methods as described herein.

[0022] Detailed description of the invention

[0023] Definitions

[0024] Within the context of the present invention, the term “in vitro" means performed or taking place in a test tube, culture dish, bioreactor or elsewhere outside a living organism.

[0025] Within the context of the present invention, the term "mammalian” identify an animal belonging to the mammalian species, for example human, cow, monkey, camel, sheep, goat etc.

[0026] Within the context of the present invention, the terms "lactocytes” or "mammary-like cells” identify secretory epithelial cells expressing CK18 cell marker and derived from mammalian induced pluripotent stem cells (miPSC) and in particular from human induced pluripotent stem cells (hiPSC). Human induced pluripotent stem cells (hiPSC) 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 current invention is e.g., hiPSC line 603, commercially available from Fujifilm Cellular Dynamics, Inc (FCDI). Further suitable hiPSCs may be selected as described e.g., in Ying Qu et al., (2017, supra). The hiPSC can be unengineered. In one embodiment, they are not engineered to comprise an exogenous nucleic acid and / or an inducible gene expression system which includes an exogenous nucleic acid, where the inducible gene expression system is configured to express a hormone or a signaling factor. In one embodiment, the exogenous nucleic acid and / or inducible gene expression system which includes an exogenous nucleic acid is promoting the cell differentiation towards lactocytes.

[0027] Within the context of the present invention the terms "mammary gland like organoids” or "mammary like organoids” mean a miniaturized and simplified version of a mammary gland which develops in two or three dimensions (2D / 3D) and which comprises lactocytes as above defined.

[0028] Within the context of the present invention, the term "human milk like product” is a cell cultured milk product. It is an edible product which is expressed by the lactocytes and / or mammary gland like organoids generated according to the process described herein.

[0029] The "human milk like product” described herein can have the same components as human breast milk of a well- nourished mother (for example in terms of bioactives, macro and micronutrients and levels thereof). This is referred to herein as a "standard human milk product”. Alternatively, "human milk like product” described herein can have altered ratios and concentrations of components found naturally in human breast milk of a well-nourished mother. This is referred to herein as a "non-standard milk like product”. A "human milk like product” described herein can be modified such that it includes components that are not found naturally in human breast milk of a well-nourished mother (a "modified milk like product”). Non-limiting examples of human milk like products are selected from the group consisting of: supplement, fortifier, human breast milk substitute (or replacer) and ingredient enriched in only one and / or a portion of bioactives, macro- and micronutrients which can be typically found in human breast milk of a well-nourished mother. The "human milk like product” can be used to replace consumption of naturally lactated milk (a "human milk substitute”). The milk substitute product can be used as a supplement (a "human milk supplement”) or as a fortifier (a "human milk fortifier”) to be consumed in combination with naturally lactated milk.

[0030] In an embodiment, the standard human milk like product described herein comprises at least macro- and micronutrients which can be typically found in human breast milk of a well-nourished mother. In one embodiment, the human milk like product described herein comprises: proteins, peptides, lipids (including linoleic acid and alpha-linolenic acid), carbohydrates, Vitamins (including Vitamin A, Vitamin D3, Vitamin E, Vitamin K, Thiamin, 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, myoinositol and L- carnitine. In one embodiment, the human milk like product described herein also comprises at least one bioactive selected from the group consisting of: growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and or exosomes), bioactives from exosome (for example miRNA) and secretory IgA. The standard human milk like product described herein is not the product of human breast lactation as occurring in nature.

[0031] The human milk like product described herein can be adapted to specific needs of the infant who will receive it. It may comprise only one and / or a portion of bioactives, macro and micronutrients which can be typically found in human breast milk of a well-nourished mother. In such embodiment, the human breast milk like product may also be referred to with the term "non-standard human milk like product”. The non-standard human milk like product described herein can comprise one or more of the 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, Thiamin, 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, myoinositol, L-carnitine, growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and or exosomes), bioactives from exosome (for example miRNA) and secretory IgA.

[0032] Within the context of the present invention, the term "non-modified human milk like product” indicates a human milk like product which is expressed by lactocytes and / or by the mammary gland like organoids generated according to steps A) and B) of the process described herein and which is not subject to the further treatment according to optional step C) of the process described herein. Non-modified human milk like product may comprise both standard and nonstandard human milk like products. Non limiting examples of non-standard human milk like products are selected from the group consisting of: supplement, fortifier, and ingredient enriched in only one and / or a portion of bioactives, macro and micro nutrients which can be typically found in human breast milk of a well-nourished mother.

[0033] Within the context of the present invention, the term "modified human milk like product” indicates a human milk like product which is expressed by lactocytes and / or by the mammary gland like organoids generated according to steps A) and B) of the process described herein and which is subject to the further treatment according to optional step C) of the process described herein. Modified human milk like products may comprise both standard and non-standard human milk like products.

[0034] Within the context of the present invention the term "EBs” means "embryoid bodies”.

[0035] Within the context of the present invention the term "mEBs” means "MammoCult medium-cultured embryoid bodies”. MammoCult medium refers to a serum-free culture medium comprising basal medium, at least one proliferation supplement, heparin and hydrocortisone.

[0036] Within the context of the present invention the terms "embryoid bodies (EBs)”, "MammoCult medium-cultured embryoid bodies (mEBs)”, "mammospheres” and / or "spheroids” refer to three-dimensional aggregates formed in suspension by pluripotent stem cells (PSC) under step A) of the process described herein.

[0037] The term "infant” in the context of the present invention identifies a child under the age of 12 months, such as under the age of 9 months, particularly under the age of 6 months.

[0038] In the context of the present invention the infant may be any term infant or preterm infant.

[0039] The term "term infant” refers to infants born at term or at a gestational age of 37 weeks or more.

[0040] The term "preterm infant” refers to infants who are born at a gestational age of less than 37 weeks.

[0041] In the context of the present invention, the term "birth weight” means the first weight of the fetus or newborn obtained after birth.

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

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

[0044] Within the context of the present invention, the term "extremely low birth weight” means a birth weight of less than 1000 g (up to and including 999 g).

[0045] The term "small for gestational age infant” refers to infants having a birth weight that is more than 2 standard deviations below the mean reference to a birth weight for gestational growth chart or having a birth weight that is less than the 10thpercentile of population-based weight data obtained from infants at the same gestational age. The term "small for gestational age infants” includes infants who are small at birth either from a constitutive or genetic origin or, as a consequence of intrauterine growth restriction.

[0046] Within the context of the present invention, the term "young children” or "toddler” indicates a child between the age of 1 and 3 years.

[0047] The term "child" as used herein refers to a person greater than three years of age up to twelve years of age.

[0048] The term "adult” as used herein refers to a person greater than twelve years of age.

[0049] Within the context of the present invention, the term "promote" and / or "promoting" as used herein refers to improving or enhancing a certain process.

[0050] Within the context of the present invention, the term "support" and / or "supporting" as used herein refer to sustaining a certain process.

[0051] The term "brain structure” as used herein refers to the structure of grey matter within the brain and specific brain regions, and in particular to synapse formation and neurite outgrowth. The term "brain connectivity” as used herein refers to the network of physical and functional connections among different regions of the brain. Structural connectivity pertains to the anatomical pathways, such as axonal projections and neural tracts, that allow communication between neurons and brain regions.

[0052] The term "intellectual potential” as used herein refers to the possible intellectual ability or capacity attainable by a subject. In particular intellectual potential may refer to fluid and / or crystallized intelligence. The term "fluid intelligence” as used herein refers to a subject's neural potential and / or a subject's novel or abstract problem solving capability. The term crystallized intelligence as used herein refers to learned or accumulated knowledge.

[0053] The term "cognitive potential” as used herein refers to the possible cognitive and / or mental ability or capacity possibly attainable by a subject. In particular the term may refer to one or more of; information processing potential, perception potential, attention potential, thinking potential, reasoning potential, understanding and remembering potential, psychomotor potential including gross motor and fine motor potential, visual potential including visual reception potential, auditory potential, language potential including expressive and receptive language potential, social emotional potential (including social potential and emotional regulation potential), memory and recall potential, concentration potential, executive function potential including problem-solving, decision-making and inhibition potential.

[0054] The term "learning potential” as used herein refers to the possible ability or capacity a subject has to learn, e.g. how easily and / or quickly a subject may be able to acquire knowledge or skills through experience, study or being taught. As well as the possible ability a subject has to adapt in response to environmental factors.

[0055] The term "cognitive functioning” as used herein refers to the intellectual processes by which one individual becomes aware of, perceives, or comprehends ideas; thus, the ability to think and understand. Cognitive functioning includes all aspects of information processing, perception, attention, thinking, reasoning, understanding and remembering as well as psychomotor, language, memory, concentration, executive functions and problem-solving abilities.

[0056] The expression "brain development" within the context of the present invention refers to brain processes and structures, selected, for example, from the group consisting of: neuronal maturation; neuronal activity, brain structure and / or brain / network connectivity.

[0057] 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 Infant Specialities (incl. Food for Special Medical Purpose) as defined in Codex Alimentarius, (Codex STAN 72-1981). It also refers to a foodstuff intended for particular nutritional use by infants during the first months of life and satisfying by itself the nutritional requirements of this category of person (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formulae and follow- on formulae). The infant formulas encompass the starter infant formulas and the follow-up or follow-on formulas. Generally, a starter formula is for infants from birth as breast-milk substitute, and a follow-up or follow-on formula from the 6th month onwards.

[0058] The "growing-up milks” (or GUMs) are given from one year onwards. It is generally a milk-based beverage adapted for the specific nutritional needs of young children. They are nutritional compositions used for feeding children from 12 months to 2-3 years old in combination with other foods.

[0059] Within the context of the present invention, the term "fortifier”” refers to a composition which comprises one or more nutrients having a nutritional benefit for infants or young children. By the term "milk fortifier”, it is meant any composition used to fortify or supplement either human breast milk, infant formula, growing-up milk or human breast milk fortified with other nutrients. Accordingly, the human milk fortifier described herein can be administered after dissolution in human breast milk, infant formula, growing-up milk or human breast milk fortified with other nutrients or otherwise it can be administered as a standalone composition.

[0060] When administered as a stand-alone composition, the human milk fortifier described herein can be also identified as being a "supplement”. In one embodiment, the milk fortifier described herein is a supplement.

[0061] By the term "human milk fortifier”, it is meant any composition used to fortify or supplement human breast milk, or human breast milk fortified with other nutrients. The "human milk fortifier” described herein can be administered to infants who were born preterm, with very low birth weight (VLBW) or with extremely low birth weight (ELBW).

[0062] The milk fortifier described herein may be in powder or liquid form.

[0063] Milk fortifier compositions having a liquid form presents some particular benefits. For example, liquid formulations might be more convenient if coupled with a packaging that delivers calibrated drops of a certain weight or volume.

[0064] In addition, liquid formulations are easier to mix with the compositions to be fortified, whereas the powder ones can, in some cases, form lumps.

[0065] Within the context of the present invention, the term "nutritional composition" means a composition which nourishes a subject. This nutritional composition is usually to be taken orally or intravenously. It may include a lipid or fat source, a carbohydrate source and / or a protein source. In a particular embodiment the nutritional composition is a ready-to-drink composition such as a ready-to-drink formula.

[0066] Within the context of the present invention, the term ‘increasing differentiation efficiency and maturation of mammalian induced pluripotent stem cell (miPSCs) into mammary-gland progenitor cells' means increasing the proportion of mammary-gland progenitor cells compared to non-mammary-gland progenitor cells, generated from a starting population of miPSCs which have undergone a differentiation protocol. Said differentiation protocol includes the use of BMP4 and RA. Thus, said increase of the proportion of mammary-gland progenitor cells may be compared to the proportion of mammary-gland progenitor cells relative to non-mammary-gland progenitor cells, generated from a starting population of miPSCs which have undergone a differentiation protocol that does not include the use of BMP4 and RA but is otherwise the same.

[0067] Within the context of the present invention, the term ‘increasing viability' means increasing the number of cells that are live and healthy. Said cells are capable of further differentiation steps, for example in the context of the present invention, are capable of developing into mammary organoids.

[0068] Within the context of the present invention, the term ‘mammary-gland progenitor cells' or similar means cells that express at least two mammary-gland progenitor markers. Said markers include but are not limited to CD49f, EpCAM, MUC1 and GAT A3. Conversely, within the context of the present invention, the term ‘non mammary-gland progenitor cells' or similar means cells that do not express at least two mammary-gland progenitor markers.

[0069] Reference herein to EpiCult or EpiCultB medium refers to a serum free culture medium comprising hydrocortisone, insulin, FGF10 and HGF.

[0070] A culture medium as disclosed anywhere herein refers to a solid, semi-solid or liquid comprising 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 methods described herein. Uses and Methods

[0071] Herein provided is an exosome, a population of exosomes, a composition comprising exosomes e.g. a nutritional or pharmaceutical composition, or an exosome product, as described anywhere herein, that may be used for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning. Also provided is a method of promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a subject in need thereof, comprising administering an exosome, a population of exosomes, a composition comprising exosomes e.g. a nutritional or pharmaceutical composition, or an exosome product, as described anywhere herein to the subject.

[0072] The exosomes described herein (and produced according to the methods described herein) have a comparable quality to exosomes isolated from in vivo produced milk and can be successfully used for increasing neuronal maturation, neuronal activity and / or network connectivity, leading to increased brain maturation, as described in Example 7. Thus, the exosomes are useful in promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning, as further demonstrated in Example 8 which describes the effects on three brain regions (cortex, hippocampus and cerebellum) observed in vivo in mice upon treatment with exosomes described herein (and produced according to the methods described herein). It is surprising that exosomes produced using an in vitro method are able to achieve these effects.

[0073] The cortex is responsible for sensory integration and processing, motor control (e.g. planning and coordination, fine motor skills, motor learning) and cognitive activities (e.g. executive functions, language processing, social cognition). Example 8 (Tables 8 and 9 and Figures 25 and 26) demonstrates that the exosomes described herein (and produced according to the methods described herein) upregulate pathways related to oligodendrocyte differentiation, myelin assembly, and ensheathment of neurons, indicative of enhanced myelination processes. Myelination is crucial for the proper functioning of neurons, as it facilitates faster signal transmission along axons. These results indicate that the exosomes described herein (and produced according to the methods described herein) can enhance cognitive abilities, improve language and communication skills, improve motor skills and coordination, and improve behaviour.

[0074] The hippocampus is responsible for learning and memories (e.g. formation, consolidation, retrieval) and spatial navigation (e.g. spatial memory, cognitive maps, path integration). The results in Example 8 (Tables 10 to 13 and Figures 27 to 30) indicate that the exosomes described herein (and produced according to the methods described herein) can enhance memory and learning due to the improvements in myelination which lead to enhanced synaptic transmission. The results also demonstrate an increased expression of genes associated with mitochondrial complexes and aerobic respiration indicating a boost in energy metabolism. Mitochondrial health is vital for neuronal survival and function, as neurons are highly energy-dependent cells. Enhanced mitochondrial function may support neuronal growth and synaptic plasticity. These results indicate that the exosomes described herein (and produced according to the methods described herein) can improve cognitive flexibility and improve cognitive overall (e.g. improving various cognitive domains such as attention, memory, and executive function).

[0075] The cerebellum is responsible for motor control (e.g. coordination of movement, balance and posture), cognitive emotional, and autonomic domains. The results in Example 8 (Tables 14 and 15 and Figures 31 and 32) indicate that the exosomes described herein (and produced according to the methods described herein) can enhance cognitive abilities due to the improved synaptic plasticity and regulation of neurotransmitter activity. The exosomes described herein (and produced according to the methods described herein) can also improve motor coordination as a result of the effects on axo-dendritic transport and cerebellar function.

[0076] Accordingly, herein provided is use of an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a healthy subject. This use can be non-therapeutic.

[0077] The exosomes described herein can be provided in a nutritional composition.

[0078] Further provided is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a subject with sub-optimal brain development. This use is therapeutic.

[0079] Sub-optimal brain development could be a subject with a neuro cognitive deficit, impairment or development delay.

[0080] In particular, the exosome, population of exosomes, composition comprising exosomes e.g. a pharmaceutical composition, or exosome product described herein may prevent, reduce the risk and / or mitigate a sub-optimal brain structure, and / or sub-optimal brain connectivity, and / or a sub-optimal intellectual potential and / or a sub-optimal cognitive potential and / or a sub-optimal learning potential and / or sub-optimal cognitive functioning in said subject.

[0081] Thus, herein provided is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in infants who are born pre-term, are small for gestational age, are low-birth weight (LBW) or who experienced intra-uterine growth retardation (IUGR) or who suffered from growth stunting because of malnutrition, such as suboptimal intra-uterine nutrition and / or disease.

[0082] Further provided is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in treating or preventing cognitive impairment in a subject.

[0083] An exosome described herein may be considered to promote or support a subject's cognitive functioning and / or intelligence if it brings one or more subjects', in particular a formula fed subject's, scores in a standardized cognitive test including intelligence test, school performance test and / or on a neurodevelopmental test, for example on the Mullen Scales of Early Learning, in line or closer to that measured or observed in a breastfed, more particularly exclusively breastfed subject, in particular of a well-nourished or nutritionally replete mother. A subject's cognitive and neurodevelopmental functioning may be considered to be in line or closer to that measured in said breastfed subject, if the difference between one or more of said subject's standardized neurodevelopmental test scores, for example Mullen's T scores, and that of said breastfed subject is less than one standard deviation, more particularly less than half a standard deviation of a standardized test score, for example less than 10 points, more particularly less than 5 points for the Mullen's T score, in particular less than 2 points. Said standardized neurodevelopmental test scores, for example Mullen's T scores, being measured at the same time point in said subject and said breastfed subject. Said Mullen's score can be measured at any appropriate time point and in particular within the first 5 years, 3 years of a human's life, more particularly the first 2 years of a human's life, even more particularly in the first year or first 6 months of a human's life.

[0084] In promoting or supporting cognitive potential, learning potential and / or intellectual potential, the exosomes described herein may have a short term or long term effect on cognitive functioning, including the development of cognitive functions, and / or learning, and on preventing or minimising any neuro cognitive deficits, impairment or delay.

[0085] Said short term effect may only be apparent in days, weeks, or months.

[0086] Said long term effect may only be apparent in years e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60. 70, 80, 90 years. The exosomes described herein (and produced according to the methods described herein) have been shown to promote key signalling pathways essential for brain function and myelination process. The exosomes described herein (and produced according to the methods described herein) have been shown to upregulate pathways related to oligodendrocyte differentiation, myelin assembly, and ensheathment of neurons indicative of enhanced myelination processes (Example 8, e.g. Tables 8 to 11 and Figures 25 to 28). Abnormalities or deficits in these processes can impact cognitive function, as seen in conditions like multiple sclerosis, where disrupted myelination produces cortical disorganization and cognitive decline.

[0087] Accordingly, provided herein is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in the treatment of a disease or condition where myelination is disrupted, e.g. a demyelinating disease. Provided herein is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in the treatment of a demyelinating disease selected from: multiple sclerosis, neuromyelitis optica spectrum disorder, transverse myelitis, acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy, and central pontine myelinolysis. In some embodiments, the demyelinating disease is multiple sclerosis.

[0088] The exosomes described herein (and produced according to the methods described herein) have been shown to promote key signalling pathways essential for promoting a robust and coordinated maturation of hippocampal circuits, facilitating higher-order cognitive functions such as learning and memory. The exosomes described herein (and produced according to the methods described herein) have been shown to promote an adaptive neurodevelopment with greater capacity for activity-dependent plasticity, resilience against neurodevelopmental disorders, and potential for cognitive flexibility. In particular, the exosomes described herein (and produced according to the methods described herein) have been shown to promote significant increase in the expression of genes included in mitochondrial pathways, improving synapse formation and neuronal connectivity (Example 8, e.g. Tables 12 and 13 and Figures 29 and 30). Accordingly, provided herein is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in the treatment of a neurodevelopmental disorder. In some embodiments, the neurodevelopmental disorder is selected from: attention- deficit / hyperactivity disorder, autism spectrum disorders, communication disorders (e.g. speech sound disorder, language disorder, stuttering, social communication disorder), intellectual disability, motor disorders (e.g. tic disorders, developmental coordination disorder, stereotypic movement disorder), and specific learning disorder. Also provided herein is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in the treatment of a neurocognitive disorder. In some embodiments, the neurocognitive disorder is selected from: Alzheimer's disease, frontotemporal lobar degeneration, Lewy body disease, vascular disease, traumatic brain injury, substance or medication use, HIV infection, prion disease, Parkinson's disease, and Huntington's disease.

[0089] The exosomes described herein (and produced according to the methods described herein) have been shown to significantly increase in the expression of genes included in synapse formation and neuropeptide signalling pathways, promoting a coordinated maturation of neuronal networks (Example 8, e.g. Tables 14 and 15 and Figures 31 and 32). Disruptions in these pathways are commonly implicated in neurodevelopmental and neuropsychiatric disorders.

[0090] Accordingly, provided herein is an exosome, a population of exosomes, a composition comprising exosomes e.g. a pharmaceutical composition, or an exosome product, as described anywhere herein, for use in the treatment of a neurodevelopmental and / or neuropsychiatric disorder. Neurodevelopmental and neuropsychiatric disorders are a group of conditions that affect brain development and function, leading to challenges in social, cognitive, and emotional functioning. In some embodiments, the neurodevelopmental disorder is selected from: attention-deficit / hyperactivity disorder, autism spectrum disorders, communication disorders (e.g. speech sound disorder, language disorder, stuttering, social communication disorder), intellectual disability, motor disorders (e.g. tic disorders, developmental coordination disorder, stereotypic movement disorder), and specific learning disorder. In some embodiments, the neuropsychiatric disorder is selected from: seizure, attention deficit disorder, cognitive deficit disorder, palsies, uncontrolled anger, migraine headaches, addictions, eating disorders, depression, and anxiety.

[0091] The exosomes described herein can be administered in a therapeutically effective amount. The exosomes described herein can be provided in a pharmaceutical composition.

[0092] The subject can be an animal. In some embodiments, the subject is a mammal. In some embodiments the subject is a human. In some embodiments the subject is an adult. In some embodiments the subject is a child. In some embodiments the subject is a young child or toddler.

[0093] In some embodiments, the subject is an infant. The subject can be an infant who are born pre-term, is small for gestational age, is low-birth weight (LBW) or has experienced intra-uterine growth retardation (IUGR) or has suffered from growth stunting because of malnutrition, such as suboptimal intra-uterine nutrition and / or disease.

[0094] In vitro Methods

[0095] Herein disclosed are methods of producing mammary gland cells using iPSCs that are cultured and differentiated in specific conditions described below and using said mammary gland cells in methods for producing a human milk like product in vitro, wherein exosomes are purified from the human milk like product in order to isolate the exosomes.

[0096] Also disclosed are methods of producing mammary gland cells using iPSCs that are cultured and differentiated in specific conditions described below and using said mammary gland cells in methods for producing a mammalian milk like product in vitro, wherein exosomes are purified from the mammalian milk like product in order to isolate the exosomes.

[0097] It has been surprisingly demonstrated that the addition of Bone Morphogenetic Protein 4 (BMP4) and / or Retinoic Acid (RA) in the methods as described anywhere herein increase the efficiency of the differentiation protocol and improve the yield and quality of the end milk like product. More specifically, it has been demonstrated that BMP4 and / or RA increase the differentiation efficiency of iPSCs to mammary gland progenitor cells, and thus increasing the number of mammary gland progenitor cells produced by the methods as described anywhere herein. The benefits of this include a higher yield of mammary gland cells. It has been demonstrated that BMP4 and RA improve expression and secretion levels of milk-specific bioactive markers such as osteopontin (OPN). It has also been demonstrated that because of the increased efficiency of differentiation due to the combination of BMP4 and RA, the overall differentiation protocol can be shortened in length. This has significant benefits including reduce cell death, higher milk like yields, and cost savings.

[0098] Mammalian mammary cell production

[0099] Methods of producing mammary gland cells using iPSCs that are cultured and differentiated in specific conditions are described herein.

[0100] A method of producing a population of mammary gland cells is described herein, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium comprising bone morphogenic protein 4 (BMP4) and / or retinoic acid (RA) to generate embryoid bodies (EBs), and ii) growing the EBs to generate a population of mammary cells.

[0101] A method of producing a population of mammary gland cells is described herein, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium comprising bone morphogenic protein 4 (BMP4) to generate embryoid bodies (EBs), and ii) growing the EBs to generate a population of mammary cells.

[0102] A method of producing a population of mammary gland cells is described herein, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium comprising retinoic acid (RA) to generate embryoid bodies (EBs), and ii) growing the EBs to generate a population of mammary cells.

[0103] A method of producing a population of mammary gland cells is described herein, comprising: i) culturing mammalian induced pluripotent stem cells (miPSCs) in a culture medium comprising bone morphogenic protein 4 (BMP4) and retinoic acid (RA) to generate embryoid bodies (EBs), and ii) growing the EBs to generate a population of mammary cells.

[0104] Use of BMP4 and / or RA for increasing the differentiation efficiency of mammalian induced pluripotent stem cell (miPSCs) into mammary-gland progenitor cells in a differentiation protocol is described herein. The use of BMP4 and / or RA for increasing the efficiency of producing a mammalian milk like product is described herein.

[0105] In some embodiments, the mammary gland cells are human mammary gland cells. In some embodiments, the mammary gland cells form lactocyte mammary-like gland organoids. Said lactocyte mammary-like gland organoids are lactogenic, i.e. are capable of producing milk.

[0106] In some embodiments, BMP4 is added to the culture medium in the early stages of the differentiation methods as described anywhere herein. In some embodiments, BMP4 is added to the culture medium between day 0 and day 10. In some embodiments, BMP4 is added to the culture medium between day 0 and day 6. In some embodiments, BMP4 is added to the culture medium between day 0 and day 3. Day 0 is the time point where the iPSCs are first added to the culture medium, i.e. when the IPSCs are first induced for differentiation.

[0107] In some embodiments, BMP4 is added to the culture medium for 3 days.

[0108] In some embodiments, BMP4 is added to the culture medium in a concentration of 5 to 20 ng / mL. In some embodiments, BMP4 is added to the culture medium in a concentration of 5 ng / mL. In some embodiments, BMP4 is added to the culture medium in a concentration of 10 ng / mL. In some embodiments, BMP4 is added to the culture medium in a concentration of 20 ng / mL.

[0109] In some embodiments, BMP4 is added to the culture medium between day 0 and day 3, and in a concentration of between 5 to 20 ng / mL.

[0110] In some embodiments, RA is added to the culture conditions in the early stages of the differentiation methods as described anywhere herein. In some embodiments, RA is added to the culture medium during the mammary lineage commitment stage. In some embodiments, RA is added to the culture medium between day 10 and day 15. In some embodiments, RA is added to the culture medium between day 6 and day 11 . In some embodiments, RA is not added to the culture medium after day 11 . In some embodiments, RA is added to the culture medium before day 11 . Day 0 is the time point where the IPSCs are first added to the culture medium, i.e. when the IPSCs are first induced for differentiation.

[0111] In some embodiments, RA is added to the culture medium for 5 days.

[0112] In some embodiments, RA is added to the culture medium in a concentration of 1 pi M.

[0113] In some embodiments, RA is added to the culture conditions between day 10 and day 15, and in a concentration of between 1 piM.

[0114] In some embodiments, BMP4 is added to the culture medium between day 0 and day 3, and RA is added to the culture medium between day 10 and day 15.

[0115] In some embodiments, BMP4 is added to the culture medium between day 0 and day 3, and RA is added to the culture medium between day 6 and day 11 .

[0116] The EBs generated in the methods as described anywhere herein express one or more mammary gland positive progenitor-cell markers. In some embodiments, said one or more mammary gland positive progenitor-cell markers are selected from EpCAM, CD49f, MUC1 and GAT A3.

[0117] In some embodiments, the EBs have increased expression of one or more mammary gland positive progenitor-cell markers compared to the expression level of said mammary gland positive progenitor-cell markers in EBs not treated with BMP4.

[0118] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EBs express one or more mammary gland 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 gland positive progenitor-cell markers. In some embodiments, said mammary gland positive progenitor-cell markers are selected from EpCAM, CD49f, MUC1 and GATA3.

[0119] In some embodiments, at least 35% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers. In some embodiments, at least 60% of EBs express EpCAM and CD49f mammary gland positive progenitorcell markers at the middle differentiation stage. In some embodiments, at least 35% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers at the pre-induction stage. In some embodiments, at least 15% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers at the post-induction stage. In some embodiments, at least 40% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers at the post-induction stage.

[0120] In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers. In some embodiments, at least 20% of EBs express MUC1 and EpCAM mammary gland positive progenitorcell markers at the middle differentiation stage. In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers at the pre-induction stage. In some embodiments, at least 5% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers at the post-induction stage. In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers at the post-induction stage.

[0121] In some embodiments, at least 20% of EBs express GATA3 and EpCAM mammary gland positive progenitor-cell markers. In some embodiments, at least 50% of EBs express GAT A3 and EpCAM mammary gland positive progenitorcell markers at the middle differentiation stage. In some embodiments, at least 25% of EBs express GAT A3 and EpCAM mammary gland positive progenitor-cell markers at the pre-induction stage. In some embodiments, at least 15% of EBs express GATA3 and EpCAM mammary gland positive progenitor-cell markers at the post-induction stage. In some embodiments, at least 40% of EBs express GAT A3 and EpCAM mammary gland positive progenitor-cell markers at the post-induction stage.

[0122] In some embodiments, the middle differentiation stage is day 25, the pre-induction stage is day 35 and the postinduction stage is day 42. In some embodiments, the middle differentiation stage is between day 20. The pre-induction stage is day 26 and the post-induction stage is day 31 .

[0123] In some embodiments, the EBs express one or more milk-specific bioactive markers. In some embodiments, said milkspecific bioactive marker is osteopontin (OPN).

[0124] In some embodiments, the EBs have increased expression of one or more milk-specific bioactive markers compared to the expression level of said markers in EBs not treated with BMP4 and / or RA. In some embodiments, the EBs have increased secretion of one or more milk-specific bioactive markers compared to the expression level of said markers in EBs not treated with BMP4 and / or RA. In some embodiments, the EBs have increased secretion of OPN compared to the expression level of OPN in EBs not treated with BMP4 and / or RA. In some embodiments, the EBs have 40% or more increased secretion of OPN compared to the expression level of OPN in EBs not treated with BMP4 and / or RA. The methods of producing a population of mammary gland cells as described anywhere herein, may be combined and / or utilized in the methods for producing a mammalian milk like product as described anywhere herein, particular as part of Step A) as described anywhere herein.

[0125] For example, in some embodiments of the method of producing a population of mammary gland cells as described anywhere herein, the culturing step i) comprises culturing the miPSCs in MammoCult medium and BMP4, in a 3D- suspension culture system, for example 3D-suspension condition, thereby directing the iPSCs to differentiate towards non-neural ectoderm cells. In some embodiments, step A i) is for at least 12 days. In some embodiments, step A i) is for no more than 8 days. In some embodiments in some embodiments of the method of producing a population of mammary gland cells as described anywhere herein, the growing step ii) comprises growing the EBs in a 3D embedding system comprising RA, for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen I. In some embodiments, step A ii) is for at least 30 days, for example for 32 days. In some embodiments, step A ii) is for at least 23 days, for example no more than 25 days.

[0126] Mammalian milk like product production

[0127] Further described are methods for producing a mammalian milk like product as defined herein, including any of steps A) and B) as defined herein and optional step C) as defined herein. Said methods for producing a mammalian milk like product as defined herein may also include the methods of producing mammalian mammary cells, as part of step A). In particular, BMP4 and / or RA may be added to any of the methods for producing a mammalian milk like product as defined herein, particularly as part of step A). Said methods also include different lengths of time for producing the mammalian milk like product.

[0128] Step A - Generating lactocytes and / or mammary like organoids from hiPSCs

[0129] According to the method described herein, mammary like cells and / or organoid structures are generated under step A).

[0130] Such mammary like cells and / or organoid structures can be generated according to any reported method making use of iPSC.

[0131] In one embodiment, such mammary like cells and / or organoid structures may be generated according to the procedure described in Ying Qu et al., Stem Cell Reports, Vol.8, 205-215 which is hereby incorporated in its entirety.

[0132] More precisely, the methodology described in the above-mentioned scientific publication (herein-below also referred to as "Ying Qu publication”, or Ying Qu, et al. (2017)) represents a two-step protocol to generate human mammary like cells and / or organoids from iPSCs.

[0133] It preferably includes as a first step (step 1) the differentiation and enrichment of non-neural ectoderm-cell-containing spheres (mEBs / mammospheres) from iPSCs and as a second step (step 2) the generation of mammary like organoids from 10-day mEBs (mammospheres) using 3D floating mixed gel-culture of Matrigel and Collagen I.

[0134] More specifically, step A preferably includes:

[0135] Embryoid bodies formation

[0136] Mammary lineage commitment

[0137] Branch and alveolar differentiation, and

[0138] Induction of milk bioactives.

[0139] Each of these stages may be conducted for a specific amount of time, with specific culture mediums.

[0140] In some embodiments, step A is conducted for a total of 40 to 45 days, preferably 42 days. In some embodiments, step A is conducted for a total of 40 to 42 days. In some embodiments, step A is conducted for 41 days

[0141] In some embodiments, step A is shortened and is conducted for less than 42 days, optionally less than 40 days, optionally less than 31 days. Preferably step A is conducted for 31 days. In some embodiments, step A is conducted for 30 to 39 days. In some embodiments, step A is conducted for 35 to 39 days. In some embodiments, step A is conducted for 30 to 35 days.

[0142] In some embodiments, the embryoid bodies formation stage is between day 0 and day 10. In some embodiments, the embryoid bodies formation stage is for 10 days.

[0143] In some embodiments, the embryoid bodies formation stage is shortened and is between day 0 and day 6. In some embodiments, the embryoid bodies formation stage is for 6 days. In some embodiments, the embryoid bodies formation stage is for 10 days or less.

[0144] In some embodiments, the mammary lineage commitment stage is between day 10 and day 15. In some embodiments, the mammary lineage commitment stage is between day 6 and day 11. In some embodiments, the embryoid bodies formation stage is for 5 days. In some embodiments, the embryoid bodies formation stage is for no more than 5 days. In some embodiments, the branch and alveolar differentiation stage is between day 15 and day 35. In some embodiments, the branch and alveolar differentiation stage is for 20 days.

[0145] In some embodiments, the branch and alveolar differentiation stage is shortened and is between day 11 and day 26. In some embodiments, the branch and alveolar differentiation stage is for 15 days. In some embodiments, the branch and alveolar differentiation stage is for no more than 15 days.

[0146] In some embodiments, the induction of milk bioactive stage is between day 35 and day 42. In some embodiments, the induction of milk bioactive stage is for 7 days. In some embodiments, the induction of milk bioactive stage is shortened and is between day 26 and day 31 . In some embodiments, the induction of milk bioactive stage is for 5 days. In some embodiments, the induction of milk bioactive stage is for no more than 5 days.

[0147] Thus, in some embodiments, step A is conducted for a total of 42 days, wherein the embryoid bodies formation stage is between day 0 and day 10, the mammary lineage commitment stage is between day 10 and day 15, the branch and alveolar differentiation stage is between day 15 and day 35, and the induction of milk bioactive stage is between day 35 and day 42.

[0148] In other embodiments, the process is shortened such that step A is conducted for a total of 31 days, wherein the embryoid bodies formation stage is between day 0 and day 6, the mammary lineage commitment stage is between day 6 and day 11 , the branch and alveolar differentiation stage is between day 11 and day 26, and the induction of milk bioactive stage is day 26 and day 31 .

[0149] Further details of the time periods and culture mediums for use in the methods described anywhere herein are provided below.

[0150] In step 1 differentiation and enrichment of non-neural ectoderm-cell-containing spheres (mEBs / mammospheres) from hiPSCs occurs by culturing hiPSCs in complete MammoCult medium (StemCell Technologies). Complete MammoCult medium is preferably composed of the basal medium, proliferation supplements, heparin (typically 4pig / mL), and hydrocortisone (typically 0.48pig / mL). Medium is usually changed every three days. mEBs (mammospheres) obtained in said step are then enriched for non-neural ectoderm cells.

[0151] In some embodiments, BMP4 is added to the culture medium in step 1.

[0152] In some embodiments, BMP4 is added to the culture medium as described in anywhere herein.

[0153] In some embodiments, BMP4 is added to the culture conditions in the early stages of the differentiation methods as described anywhere herein. In some embodiments, BMP4 is added to the culture medium between day 0 and day 10. In some embodiments, BMP4 is added to the culture medium between day 0 and day 6. In some embodiments, BMP4 is added to the culture medium between day 0 and day 3. Day 0 is the time point where the iPSCs are first added to the culture medium, i.e. when the iPSCs are first induced for differentiation.

[0154] In some embodiments, BMP4 is added to the culture medium for 3 days.

[0155] In some embodiments, BMP4 is added to the culture medium in a concentration of 5 to 20 ng / mL. In some embodiments, BMP4 is added to the culture medium in a concentration of 5 ng / mL. In some embodiments, BMP4 is added to the culture medium in a concentration of 10 ng / mL. In some embodiments, BMP4 is added to the culture medium in a concentration of 20 ng / mL.

[0156] In some embodiments, BMP4 is added to the culture medium between day 0 and day 3, and in a concentration of between 5 to 20 ng / mL.

[0157] In step 2, following the protocol of Ying Qu, et al. (2017), mammary-like organoids are generated by firstly preparing a 3D culture on basis of a floating mixed gel (e.g. Matrigel and Collagen I). 10 day mEBs (mammospheres) are then grown for 5 days in the mixed gel floated in complete EpiCultB medium supplemented with Parathyroid hormone (pTHrP). For induction of branch and alveolar differentiation for preparation of mammary like organoids / lactocytes, cells are then cultured in complete EpiCultB medium supplemented with hydrocortisone, insulin, FGF10 and HGF. Milk protein expression is typically induced at day 35 by adding prolactin, hydrocortisone and insulin to complete EpiCultB medium supplemented with BSA (lactogenic medium) and culturing for 5 days. The process of Ying Qu, et al. (2017) is typically completed at day 40.

[0158] In some embodiments, RA is added to the culture medium in step 2.

[0159] In some embodiments, RA is added to the culture medium as described in anywhere herein.

[0160] In some embodiments, RA is added to the culture medium in the early stages of the differentiation methods as described anywhere herein. In some embodiments, RA is added to the culture medium during the mammary lineage commitment stage. In some embodiments, RA is added to the culture medium between day 10 and day 15. In some embodiments, RA is added to the culture medium between day 6 and day 11. In some embodiments, RA is not added to the culture medium after day 11 . Day 0 is the time point where the iPSCs are first added to the culture medium, i.e. when the iPSCs are first induced for differentiation.

[0161] In some embodiments, RA is added to the culture medium for 5 days.

[0162] In some embodiments, RA is added to the culture medium in a concentration of 1 pi M.

[0163] In some embodiments, RA is added to the culture medium between day 10 and day 15, and in a concentration of between 1 piM.

[0164] Also described herein is a method for producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises: i) directing iPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, and after 10 days collecting mammospheres formed thereof; and ii) growing such mammospheres in an appropriate system comprising RA as described anywhere herein (for example a floating mixed gel culture system as described in Hassiotou F. et al. Stem Cells. 2012) for at least 10 days to generate lactocytes. Also described herein is a method for producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises: i) directing iPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, and after 6 days collecting mammospheres formed thereof; and ii) growing such mammospheres in an appropriate system comprising RA as described anywhere herein (for example a floating mixed gel culture system as described in Hassiotou F. et al. Stem Cells. 2012) for less than 10 days to generate lactocytes.

[0165] In another embodiment, a method for producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises: i) directing iPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, in non-adherent conditions for mammospheres formation; and ii) growing such mammospheres in a 3D appropriate system comprising RA as described anywhere herein (for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen or in suspension cultures in nonadherent plates) for at least 10 days to generate lactocytes.

[0166] In another embodiment, a method for producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises: i) directing iPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, in non-adherent conditions for mammospheres formation; and ii) growing such mammospheres in a 3D appropriate system comprising RA as described anywhere herein (for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen or in suspension cultures in non- adherent plates) for less than 10 days to generate lactocytes.

[0167] In one embodiment, mammary commitment under step A) is obtained by applying a conditioned medium (for example EpiCultB) supplemented with specific factors (for example Parathyroid hormone (pTHrP), hydrocortisone, insulin, FGF10, and HGF) and RA.

[0168] The method described herein can further comprise generating mammary - like organoids under step A).

[0169] The method to generate mammary - like organoids under step A) can include culturing the cells under conditions selected from the group consisting of: 2D monolayers of cells, 2D with attached EBs, in suspension in non-adherent plates and in mixed floating gel.

[0170] In a preferred embodiment, mixed floating gel comprises Matrigel and Collagen I.

[0171] In another preferred embodiment, mammospheres (mEBs) in step A) are grown in an appropriate system (for example a floating mixed gel culture system as described in Hassiotou F. et al. Stem Cells. 2012) for at least 15 days.

[0172] In a more preferred embodiment, mammospheres (mEBs) in step A) are grown in an appropriate system (for example a floating mixed gel culture system as described in Hassiotou F. et al. Stem Cells. 2012) for 20 days.

[0173] In one embodiment, the method described herein provides for culture conditions according to step A) [for example under step A)i) and / or under step A)ii)], which are adapted to generate lactocytes derived from human induced pluripotent stem cells (hiPSC) capable to secrete a human milk like product.

[0174] In a preferred embodiment, a method for producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises directing hiPSCs to differentiate towards mammary gland cells (for example lactocytes) in an appropriate 3D culture system comprising BMP4 and / or RA as described anywhere herein (for example 3D-suspension condition). In some embodiments, for at least 42 days. In some embodiments, for no more than 31 days. In some embodiments, for at least 31 days.

[0175] In another preferred embodiment, a method for producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises:

[0176] I) directing hiPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, in an appropriate 3D culture system (for example 3D-suspension condition) for at least 12 days (day -2 to day 10), and

[0177] II) growing the formed mEBs (mammospheres) in an appropriate 3D embedding system comprising RA as described anywhere herein (for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen I) for at least 30 days, preferably for 32 days, to generate lactocytes.

[0178] In another preferred embodiment, a method for producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises:

[0179] I) directing hiPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, in an appropriate 3D culture system (for example 3D-suspension condition) for no more than 8 days (day -2 to day 6), and

[0180] II) growing the formed mEBs (mammospheres) in an appropriate 3D embedding system comprising RA as described anywhere herein (for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen I) for no more than 25 days, to generate lactocytes.

[0181] A method of producing a human milk like product is described herein comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSCs), wherein step A)i) is defined as follows:

[0182] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 (comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL as described in Chen et al., Nat Methods, 2011) or mTeSR™ for two days (day -2-day 0), and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in complete MammoCult medium (StemCell Technologies) comprising the basal medium, proliferation supplement and supplemented with heparin (typically 4pig / mL), hydrocortisone (typically 0.48pig / mL) and BMP4 as described anywhere herein, for 10 days (day 0-day 10), and wherein step A)ii) is distinguished into further substeps and comprises the following steps: II), ill) and iv):

[0183] II) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) for 5 days (day 10-day 15), iii) promotion of branch and alveolar differentiation and mammary cell specification by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 20 days (day 15-day 35), and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 7 days (day 35-day 42).

[0184] Further described is a method of producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSCs), wherein step A)i) is defined as follows:

[0185] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 (comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL as described in Chen et al., Nat Methods, 2011) or mTeSR™ for two days (day -2-day 0), and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in complete MammoCult medium (StemCell Technologies) comprising the basal medium, proliferation supplement and supplemented with heparin (typically 4pig / mL), hydrocortisone (typically 0.48pig / mL) and BMP4 as described anywhere herein, for 6 days (day 0-day 6), and wherein step A)ii) is distinguished into further substeps and comprises the following steps: ii), iii) and iv): ii) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) and RA for 5 days (day 6-day 11), iii) promotion of branch and alveolar differentiation and mammary cell specification by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 15 days (day 11 -day 26), and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 5 days (day 26-day 31).

[0186] Step iv) preferably leads to differentiation into milk protein expressing cells, particularly lactocytes, and / or mammary like gland organoids.

[0187] Further described is a method of producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSCs), wherein step A)i) is defined as follows:

[0188] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 (comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL as described in Chen et al., Nat Methods, 2011) mTeSR™ for two days (day-2-day 0), and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin and BMP4 as described anywhere herein, for 10 days (day 0-day 10), and wherein step A)ii) is distinguished into further substeps and comprises the following steps ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I floated in EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) and RA for 5 days (day 10-day 15), iii) promotion of branch and alveolar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 20 days (day 15 to day 35), and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 7 days (day 35 to day 42).

[0189] Further described is a method of producing a human milk like product is provided comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSCs), wherein step A)i) is defined as follows:

[0190] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 (comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL as described in Chen et al., Nat Methods, 2011) mTeSR™ for two days (day-2-day 0), and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin and BMP4 as described anywhere herein, for 6 days (day 0-day 6), and wherein step A)ii) is distinguished into further substeps and comprises the following steps ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I floated in EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) and RA for 5 days (day 6-day

[0191] I I), iii) promotion of branch and alveolar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGFIO and HGF for 15 days (day 11 to day 26), and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 5 days (day 26 to day 31).

[0192] Step iv) preferably leads to differentiation into milk protein expressing cells, particularly lactocytes, and / or mammary like gland organoids.

[0193] Standard IPSC medium E8 (comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCO3 and transferrin, TGFpl or NODAL as described in Chen et al., Nat Methods, 2011) as mentioned herein is commercially available, e.g., as "Essential 8™ Medium” from ThermoFischer Scientific, catalogue number A1517001 (see also https: / / www.thermofisher.com / order / cataloq / product / A1517001 # / A1517001 ). mTeSR™ medium is commercially available from STEMCELL Technologies, catalogue number 85850 (see also https: / / www.stemcell.com / mtesr1.html). Such medium is also described in "Defined, Feeder- Independent medium for human hembryonic stem cell culture”, Current protocol in Stem Cell Biology, Volume 2, Issue 1 , Sept 2007.

[0194] In one embodiment, steps iii) and / or iv) as defined above for the particularly preferred embodiments, preferably lead to formation of / differentiation into at least breast cells, luminal cells, and basal cells. In this context, breast cells preferably express one or more, preferably all of markers selected from the group consisting of: p-Casein, milk protein, and hormone receptors. Moreover, luminal cells preferably express one or more, preferably all markers selected from the group consisting of: EpCAM, MUC1 , CD49F, GAT A3, CK8, and CK18. Moreover, basal cells preferably express one or more markers selected from the group consisting of: CK14, o-smooth muscle actin and P63.

[0195] In one further embodiment, after induction of mEBs (mammospheres) in step II) and / or iv) as defined above for the particularly preferred embodiments, mammary like gland organoids may be obtained, that express one or more markers selected from the group consisting of: p-Casein, milk protein, and hormone receptors, luminal cells that express one or more markers selected from the group consisting of: EpCAM, MUC1 , CD49F, GATA3, CK8, CK18, and basal cells that express one or more markers selected from the group consisting of: CK14, o-smooth muscle actin and P63.

[0196] The methods described above are provided for producing a human milk like product.

[0197] In one embodiment (of step A), delivery of nutrients and biomimetic stimuli is controlled to influence cell growth, differentiation and tissue formation. In one embodiment (of step A), such control is performed in a bioreactor.

[0198] Where BMP4 and / or RA are added to the culture medium in the methods for producing a mammalian milk like product as described anywhere herein, the EBs generated in the methods as described anywhere herein express one or more mammary gland positive progenitor-cell markers. In some embodiments, said one or more mammary gland positive progenitor-cell markers are selected from EpCAM, CD49f, MUC1 and GAT A3.

[0199] In some embodiments, the EBs have increased expression of one or more mammary gland positive progenitor-cell markers compared to the expression level of said mammary gland positive progenitor-cell markers in EBs not treated with BMP4.

[0200] In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EBs express one or more mammary gland 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 gland positive progenitor-cell markers. In some embodiments, said mammary gland positive progenitor-cell markers are selected from EpCAM, CD49f, MUC1 and GATA3.

[0201] In some embodiments, at least 35% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers. In some embodiments, at least 60% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers at the middle differentiation stage. In some embodiments, at least 35% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers at the pre-induction stage. In some embodiments, at least 40% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers at the post-induction stage.

[0202] In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers. In some embodiments, at least 20% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers at the middle differentiation stage. In some embodiments, at least 15% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers at the pre-induction stage. In some embodiments, at least 5% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers at the post-induction stage.

[0203] In some embodiments, at least 20% of EBs express GAT A3 and EpCAM mammary gland positive progenitor-cell markers. In some embodiments, at least 50% of EBs express GAT A3 and EpCAM mammary gland positive progenitor-cell markers at the middle differentiation stage. In some embodiments, at least 25% of EBs express GAT A3 and EpCAM mammary gland positive progenitor-cell markers at the pre-induction stage. In some embodiments, at least 15% of EBs express GAT A3 and EpCAM mammary gland positive progenitor-cell markers at the post-induction stage.

[0204] In some embodiments, the middle differentiation stage is day 25. The pre-induction stage is day 35 and the postinduction stage is day 42. In some embodiments, the middle differentiation stage is between day 20. The pre-induction stage is day 26 and the post-induction stage is day 31 .

[0205] In some embodiments, the EBs express one or more milk-specific bioactive markers. In some embodiments, said milkspecific bioactive marker is osteopontin (OPN).

[0206] In some embodiments, the EBs have increased expression of one or more milk-specific bioactive markers compared to the expression level of said non-neuronal ectodermal markers in EBs not treated with BMP4 and / or RA.

[0207] Thus, as an example, provided herein is a method for producing a human milk like product, comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises:

[0208] I) directing IPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, and after 10 days collecting mammospheres formed thereof; and ii) growing such mammospheres in an appropriate system (for example a floating mixed gel culture system as described in Hassiotou F. et al. Stem Cells. 2012) for at least 10 days to generate lactocytes, wherein the mammospheres (EBs) have increased expression of one or more mammary gland positive progenitor-cell markers compared to the expression level of said mammary gland positive progenitor-cell markers in mammospheres (EBs) not treated with BMP4.

[0209] Thus, as an example, provided herein is a method for producing a human milk like product, comprising generating lactocytes under step A) from human induced pluripotent stem cells (hiPSC), where such step A) comprises:

[0210] I) directing IPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium (for example MammoCult medium) and BMP4 as described anywhere herein, and after 6 days collecting mammospheres formed thereof; and

[0211] II) growing such mammospheres in an appropriate system comprising RA (for example a floating mixed gel culture system as described in Hassiotou F. et al. Stem Cells. 2012) for less than 10 days to generate lactocytes, wherein the mammospheres (EBs) have increased expression of one or more mammary gland positive progenitorcell markers compared to the expression level of said mammary gland positive progenitor-cell markers in mammospheres (EBs) not treated with BMP4 and / or RA.

[0212] It will be understood that any method or method step disclosed herein may be conducted in 3D suspension culture rather than using a membrane matrix as a support. Thus, in some embodiments, during all the differentiation procedure, cells are maintained in suspension culture.

[0213] Step B - Expressing a human breast milk like product

[0214] The methods described herein can comprise expressing the human milk like product from mammary like organoids derived from human induced pluripotent stem cells (hiPSCs), preferably prepared according to step A). Expressing human milk like products preferably occurs upon induction of expression of the human milk like product from such lactocytes and / or mammary-like gland organoids. In one embodiment, lactating lactocytes are induced by applying a specific medium (for example EpiCultB) supplemented with lactogenic factors (for example prolactin, hydrocortisone, and insulin).

[0215] Particularly, 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 bioactives of human milk, selected from the group comprising or consisting of proteins, lipids or oligosaccharides, preferably human milk oligosaccharides, etc. Inventors particularly managed to identify with the particularly preferred protocol according to steps A i) to iv) as carried out above inter alia oligosaccharides (including lactose and some HMOs), lipids (including 4 fatty acids), proteins (7 detected including caseins), and miRNA (75 detected, including 11 typically detected in HBM).

[0216] 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 bioactives of human milk, selected from the group comprising or consisting of: oligosaccharides, lipids, proteins, exosomes and miRNA. 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 exosomes.

[0217] In another embodiment, human milk like product obtained from mammary like organoids derived from human induced pluripotent stem cells (hiPSCs), preferably prepared according to step A), contains bioactives of human milk, selected from the group comprising or 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: 1 n7 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 n3 fatty acid, C-22:0 fatty acid, lactoferrin, albumin, prolactin, Alpha S1 -casein, Hemoglobin subunit beta, Hemoglobin subunit alpha, a- lactalbumin, Alpha-2-macroglobulin, p-casein, bile salt-activated lipase, K-casein, lactadherin, CD14, fatty acid synthase, IgA, plgR, Serum albumin, Xanthine dehydrogenase, exosomes, miR-21-5p, miR-181 a-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.

[0218] The human milk like product obtained from mammary like organoids derived human induced pluripotent stem cells (hiPSCs) can be a standard human milk product. In another embodiment of the present invention, the human milk like product obtained from mammary like organoids derived from human induced pluripotent stem cells (hiPSCs), is a non -standard human milk product.

[0219] Step C - Further treatments to produce modified human breast milk like product

[0220] The herein-described methods can comprise an additional step C) which is performed on the human milk like product obtainable from step B) and which comprises performing an additional treatment on such product to provide a modified human milk like product.

[0221] In one particular embodiment, the additional treatment step C) performed on the inventive human breast milk like product 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 (for example which can't be expressed by the human mammary gland organoid (such as for example Immunoglobulins, probiotic and / or minerals) or combinations thereof.

[0222] In particular, in some embodiments, the human milk like product described anywhere herein is extracted, isolated and purified as part of the methods described anywhere herein. Thus, the invention provides an isolated human milk like product. In some embodiments, the mammalian milk like product described anywhere herein is extracted, isolated and purified as part of the methods described anywhere herein. Thus, the invention provides an isolated mammalian milk like product, for example an isolated human milk like product.

[0223] In some embodiments, the isolated human milk like product is formulated into a composition. In some embodiments, the isolated human milk like product is formulated into a nutritional composition. In some embodiments, the isolated human milk like product is formulated into a pharmaceutical composition.

[0224] In some embodiments, the isolated mammalian milk like product is formulated into a composition. In some embodiments, the isolated mammalian milk like product is formulated into a nutritional composition. In some embodiments, the isolated mammalian milk like product is formulated into a pharmaceutical composition.

[0225] In some embodiments, particular bioactives of the human milk like product, selected from the group comprising or consisting of: oligosaccharides, lipids, proteins, exosomes and miRNA, are extracted, isolated and purified from the human milk like product. In particular, exosomes are extracted, isolated and purified from the human milk like product. Thus, the invention provides isolated bioactives of the human milk like product, selected from the group comprising or consisting of: oligosaccharides, lipids, proteins, exosomes and miRNA. In particular, the invention provides isolated exosomes.

[0226] In some embodiments, particular bioactives of the mammalian milk like product, selected from the group comprising or consisting of: oligosaccharides, lipids, proteins, exosomes and miRNA, are extracted, isolated and purified from the mammalian milk like product. In particular, exosomes are extracted, isolated and purified from the mammalian milk like product. Thus, the invention provides isolated bioactives of the mammalian milk like product, selected from the group comprising or consisting of: oligosaccharides, lipids, proteins, exosomes and miRNA.

[0227] In particular, exosomes can be extracted, isolated and purified from the mammalian milk like product, for example human exosomes can be extracted, isolated and purified from the human milk like product.

[0228] In some embodiments, the isolated bioactives of the human milk like product are formulated into a composition. In some embodiments, the isolated bioactives of the human milk like product are formulated into a nutritional composition. In some embodiments, the isolated bioactives of the human milk like product are formulated into a pharmaceutical composition.

[0229] In some embodiments, the isolated bioactives of the mammalian milk like product are formulated into a composition. In some embodiments, the isolated bioactives of the mammalian milk like product are formulated into a nutritional composition. In some embodiments, the isolated bioactives of the mammalian milk like product are formulated into a pharmaceutical composition.

[0230] In some embodiments, the isolated exosomes of the human milk like product are formulated into a composition. In some embodiments, the isolated exosomes of the human milk like product are formulated into a nutritional composition. In some embodiments, the isolated exosomes of the human milk like product are formulated into a pharmaceutical composition.

[0231] In some embodiments, the isolated exosomes of the mammalian milk like product are formulated into a composition. In some embodiments, the isolated exosomes of the mammalian milk like product are formulated into a nutritional composition. In some embodiments, the isolated exosomes of the mammalian milk like product are formulated into a pharmaceutical composition. In some embodiments, step C) comprises purifying exosomes from the human milk like product. In some embodiments, step C) comprises purifying exosomes from the human milk like product to remove impurities, for example by chromatography or filtration or ultracentrifugation, in order to isolate the exosomes. In some embodiments, step C) comprises purifying exosomes from the human milk like product by chromatography in order to isolate the exosomes. In some embodiments, step C) comprises purifying exosomes from the human milk like product by filtration in order to isolate the exosomes. In some embodiments, step C) comprises purifying exosomes from the human milk like product by ultracentrifugation in order to isolate the exosomes.

[0232] In some embodiments, the chromatography is column-based chromatography, for example size inclusion or size exclusion chromatography.

[0233] It will be understood that a product obtained from a purified compared to a non-purified protocol will be different in terms of its composition. In particular, the surface composition of the product will be different as components such as proteins that are attached to the surface of a product molecule may be removed during the purification steps. For example, proteins present on the surface of the exosomes may be removed.

[0234] In some embodiments, step C) further comprises sterilising the isolated exosomes.

[0235] In some embodiments, step C) further comprises storing the isolated exosomes at below freezing, optionally at -80°C. In some embodiments, step C) comprises formulating the isolated exosomes into a powder form, for example by freeze drying or spray drying, or a liquid form.

[0236] In some embodiments, the method further comprises formulating the isolated exosomes with supplementary nutritional ingredients.

[0237] In some embodiments, the method further comprises dispensing the isolated exosomes into a container for consumption.

[0238] Human milk like products

[0239] ‘Standard’ human breast milk like product

[0240] The human breast milk like product can be a 'standard' human breast milk like product, i.e., comprises the same components as human breast milk of a well-nourished mother.

[0241] The benefits of breast feeding are well known in the scientific literature and the possibility to have access to human breast milk like product allows its use for a number of equally well-known health benefits.

[0242] In such an embodiment, the human breast milk like product can be used as a substitute of breastfeeding under circumstances where real breastfeeding is not possible.

[0243] In such embodiment, the human breast milk like product is intended to be used for example to support longer breastfeeding experience for women who have less milk or who stop to produce milk after 6 months from birth.

[0244] Similarly, the human breast milk like product is intended to be used for example to allow breastfeeding even under circumstances where sicknesses compromise real breastfeeding from the mother.

[0245] In another embodiment, the human breast milk like product is intended to be used under circumstances whereby breastmilk production would not naturally be initiated, for example if an infant is adopted.

[0246] In one embodiment, the human breast milk like product is a non-modified human breast milk like product.

[0247] In another embodiment, the human breast milk like product is a modified human breast milk like product. In one embodiment, the human milk like product according to the present invention comprises: proteins, lipids, carbohydrates, vitamins and minerals.

[0248] In another embodiment, the human milk like product according to the present invention comprises: proteins, lipids, carbohydrates, vitamins, minerals and bioactives.

[0249] In one embodiment, the human milk like product according to the present invention comprises: proteins, lipids (including linoleic acid and alpha-linolenic acid), carbohydrates, Vitamins (including Vitamin A, Vitamin D3, Vitamin E, Vitamin K, Thiamin, 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, myoinositol and L-carnitine.

[0250] In a further embodiment, the human milk like product according to the present invention also comprises at least one bioactive selected in the group consisting of: growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and or exosomes), bioactives from exosomes (for example miRNA) and secretory IgA.

[0251] Such human breast milk like product may be prepared according to the method of the present invention for example by including a step C) of addition of growth factors, cytokines, probiotics, extracellular vesicles (e.g. milk fat globules and or exosomes), bioactives from exosomes (for example miRNA) and secretory IgA.

[0252] In one embodiment, the human breast milk like product contains probiotics.

[0253] Such human breast milk like product may be prepared according to the method of the present invention for example by including a step C) of addition of probiotics (for example B.Lactis, B.lnfantis, L. Ramnhosus) which can be obtained from several commercially available sources.

[0254] In such embodiment, the human breast milk like product may be used for optimizing gastro intestinal function and / or promoting Immunity.

[0255] In one embodiment, the human breast milk like product contains secretory IgA and probiotics.

[0256] Such human breast milk like product may be prepared according to the method described herein for example by including a step C) of addition of a combination of probiotics and secretory IgA which may be prepared as described for example in patent applications W02009 / 156301 and W02009 / 156367 which are hereby incorporated by reference.

[0257] ‘Non-standard’ human breast milk like product

[0258] In one embodiment of the present invention, the human milk like product can have altered ratios and concentrations of components found naturally in human breast milk of a well-nourished mother. This is referred to herein as a "nonstandard milk like product”.

[0259] In one embodiment, the human milk like product according to the present invention may be selected from the group consisting of a milk fortifier, a supplement, and / or a human breast milk replacer adapted for special purposes.

[0260] Human Milk fortifiers and Human milk bioactive supplements

[0261] In one embodiment, the method described herein provides for a human breast milk like product which may be used to fortify human breast milk naturally obtained from a nursing mother or to fortify infant formulas.

[0262] In another embodiment, the method described herein provides for a human breast milk like product which may be used as a supplement for infants or young children in need thereof. Remarkably, the human origin of the constituents (especially bioactive constituents) in such fortifiers or supplements combined with the fact that they are according to the method described herein, is supposed to provide to such constituents an intact or higher functionality.

[0263] The human breast milk like product is preferably intended to be used as a fortifier. Such human breast milk like product intended to be used as a fortifier and may be prepared according to the method described herein for example by including a step C) of isolation and / or enrichment of (certain) bioactives from the human breast milk like product obtainable from step B). Such isolation step may be performed via classical fractionation, enrichment and / or purification of the non-modified human breast milk like product obtainable from step B).

[0264] The human breast milk like product intended to be used as a supplement may comprise one or more bioactives selected from the group consisting of: human milk oligosaccharides (for example 2FL, 3FL, LNT, LnNT, DiFI, 6SL and / or 3SL), lipids, growth factors (for example epidermal growth factor (EGF), heparin binding epidermal growth factor), cytokines (for example transforming growth factor -beta 2 (TGFbeta-2), IL-1. IL-2, IL-6, IL-10, IL-18, interferon gamma (INF- gamma), TNF-alpha), extracellular vesicles (e.g. milk fat globules and or exosomes), exosome comprising microRNAs and antimicrobial / protecting bioactives (for example IgA, lactoferrin, lysozyme, lactadherine). Such human breast milk like product intended to be used as a supplement may be prepared according to the method of the present invention for example by including a step C) of isolation of the bioactives from the non-modified human breast milk like product obtainable from step B). Such isolation step may be performed via classical fractionation, enrichment and / or purification of the non -modified human breast milk like product obtainable from step B).

[0265] In one embodiment, the human breast milk like product is a supplement or milk fortifier which contains fucosylated human milk oligosaccharides, for example 2FL and / or 3FL. Such supplement or milk fortifier is for use in completing the profile of human breast milk of women who do not secrete fucosylated oligosaccharides because of the inactivity of their FUT2 gene.

[0266] Such human breast milk like product intended to be used as a fortifier or supplement may be prepared according to the method described herein for example by including a step C) of isolation and / or enrichment of fucosylated oligosaccharides (for example 2FL and or 3FL) from the non-modified human breast milk like product obtainable from step B).

[0267] Components of human milk like products

[0268] As previously described herein, the human milk like product described herein comprises at least one bioactive. One such bioactive is exosomes.

[0269] Exosomes

[0270] Human breast milk exosomes are small microvesicles involved in infant development, for example in immunity, metabolism and development. They are thought to play an important role in therapy and disease prevention.

[0271] The exosomes comprise a lipid bilayer membrane that encapsulates a combination of molecules including proteins, lipids, messengerRNA (mRNA) and microRNA (miRNA). The types and concentrations of these molecules determine the exosome's fingerprint and function.

[0272] The present invention provides for uses of isolated human breast milk exosomes. The exosomes of described herein possess the same lipid bilayer vesicle structure and function to that of a naturally derived human breast milk exosome. The exosomes described herein are not produced from naturally derived human breast milk.

[0273] The exosomes described herein can be isolated from the human milk like product described anywhere herein, wherein the human milk like product is produced from any method described herein.

[0274] Thus, for example, the exosomes can be produced by an in vitro method, the method comprising:

[0275] A) Generating lactocyte mammary-like gland organoids derived from human induced pluripotent stem cells (hiPSC)

[0276] B) Secreting a human milk like product from said lactocytes, and

[0277] C) Purifying the exosomes from the human milk like product by chromatography or filtration or ultracentrifugation in order to isolate the exosomes, wherein step A) comprises culturing the hiPSCs in a culture medium comprising BMP4 and / or RA, optionally, wherein step A) is conducted for a total of 42 days or 31 days, and / or wherein step A) is conducted in 3D-suspension conditions.

[0278] The exosomes described herein differ from exosomes derived from natural human breast milk by their composition.

[0279] In some embodiments, the exosomes of the present invention differ from exosomes derived from natural human breast milk by their proteome profile. It will be understood that a proteome profile relates to the types of proteins present in the exosomes and / or the concentration of the proteins present in the exosomes.

[0280] In some embodiments, the exosomes of the present invention are substantially the same as exosomes derived from natural human breast milk by one or more characteristics selected from: size, miRNA profile and / or lipid profile. In some embodiments, the size, miRNA profile and lipid profile of the exosomes of the present invention are substantially the same as exosomes derived from natural human breast milk.

[0281] In some embodiments, differences in the proteome profile is the only difference between exosome of the present invention and exosomes derived from natural human breast milk.

[0282] The present invention also relates to a population of exosomes as described anywhere herein. In some embodiments, the exosomes in said population are identical or different in terms of their structure, function and / or composition. In some embodiments, the exosomes in said population are identical. In some embodiments, the exosomes in said population differ by their composition.

[0283] In some embodiments, the exosome or population of exosomes as described herein are formulated in a powder form, for example by freeze drying or spray drying, or a liquid form. In some embodiments, the exosome or population of exosomes as described herein are frozen, optionally at -80°C. In some embodiments, the exosome or population of exosomes as described herein are sterilised.

[0284] In some embodiments, the exosome or population of exosomes as described herein are packaged into a container for consumption.

[0285] In some embodiments, the present invention relates to a composition comprising the exosomes or population of exosomes as described anywhere herein. In some embodiments, the present invention relates to a nutritional composition comprising the exosomes or population of exosomes as described anywhere herein.

[0286] In some embodiments, the present invention provides a composition comprising the exosome or population of exosomes as described anywhere herein. In some embodiments, the composition is a nutritional composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises supplementary nutritional ingredients.

[0287] In some embodiments, the present invention provides a pharmaceutical composition comprising the exosome or population of exosomes as described anywhere herein and a pharmaceutically acceptable excipient.

[0288] The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0289] In some embodiments, the composition as described anywhere herein (e.g. the nutritional or pharmaceutical composition) comprises exosomes that have been isolated from the human milk like product described anywhere herein, wherein the human milk like product is produced from any method described herein.

[0290] Thus, for example, the composition (e.g. a nutritional or pharmaceutical composition) comprising exosomes for the uses and methods described herein can be produced according to an in vitro method comprising:

[0291] A) Generating lactocyte mammary-like gland organoids derived from human induced pluripotent stem cells (hiPSC)

[0292] B) Secreting a human milk like product from said lactocytes, and

[0293] C) Purifying the exosomes from the human milk like product by chromatography or filtration or ultracentrifugation in order to isolate the exosomes, wherein step A) comprises culturing the hiPSCs in a culture medium comprising BMP4 and / or RA, optionally, wherein step A) is conducted for a total of 42 days or 31 days, and / or wherein step A) is conducted in 3D-suspension conditions.

[0294] In some embodiments, the composition as described anywhere herein is formulated into a powder form, for example by freeze drying or spray drying, or a liquid form. In some embodiments, the composition as described anywhere herein is frozen, optionally at -80°C.

[0295] In some embodiments, the composition as described anywhere herein is packaged into a container for consumption.

[0296] Exosome proteomics

[0297] Through proteomics analysis, about 2000 proteins have been identified in human breast milk exosomes in the literature. Proteomics analysis of the exosomes described herein surprisingly demonstrated that the exosomes possess similar but not identical proteomes to those of exosomes derived from natural human breast milk.

[0298] In particular, the exosomes described herein were found to be missing one or more proteins that are present in exosomes derived from natural human breast. In some embodiments, these proteins include but are not limited to Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1). In some embodiments, these proteins include but are not limited to C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0299] In some embodiments, Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagenlysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in an isolated human breast milk exosome of the invention. In some embodiments, Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are detected by liquid chromatography - mass spectrometry conducted under the same conditions in an exosome isolated from human breast milk.

[0300] In some embodiments, C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are not detected by liquid chromatography - mass spectrometry in an isolated human breast milk exosome of the invention. In some embodiments, C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are detected by liquid chromatography - mass spectrometry conducted under the same conditions in an exosome isolated from human breast milk.

[0301] In the present invention, detection of the exosome protein profile was assayed by liquid chromatography - mass spectrometry (LC-MS). The sensitivity of LC / MS analysis is at the femtomole (fmol) level.

[0302] Exemplary, but not limiting, suitable mass spectrometers include Orbitrap Fusion™ Lumos™ Tribrid™. Exemplary, but not limiting, suitable HPLC systems include Vanquish™ Neo UHPLC System, 300 nL / min, and exemplary, but not limiting, suitable HPLC column include Acclaim™ PepMap™ RSLC C18 2pim 50 cmx75 pm. Overall HPLC gradient time is, for example, about 100 min to about 200 min, preferably about 180 min. In some embodiments, the overall HPLC gradient time is 180 min.

[0303] In another embodiment, the mass spectrometer is Orbitrap Fusion™ Lumos™ Tribrid™, the HPLC system is Vanquish™ Neo UHPLC System, 300 nL / min, and the HPLC column is Acclaim™ PepMap™ RSLC C18 2pim 50 cmx75 pm. In some embodiments, the mass spectrometer is Orbitrap Fusion™ Lumos™ Tribrid™, the HPLC system is Vanquish™ Neo UHPLC System, 300 nL / min, and the HPLC column is Acclaim™ PepMap™ RSLC C18 2pim 50 cmx75 pm using an overall HPLC gradient time of 180 min.

[0304] The amount of proteins used to prepare the samples for proteomics LC / MS analysis is about 15 pig to about 50 pig, preferably about 20 pig to about 35 pig, more preferably about 25 pig. The amount of peptides injected per LC MS / MS analysis (i.e., on column) is about 0.5 pig to about 5 pig, preferably about 1 pig to about 5 pig, more preferable about 1 pig to about 2.5 pig, In some embodiments, about 1 pig of proteins used to prepare the samples for proteomics LC / MS analysis. In other embodiments, about 2.5 pig of proteins used to prepare the samples for proteomics LC / MS analysis. In some embodiments, the mass spectrometer is Orbitrap Fusion™ Lumos™ Tribrid™, the HPLC system is Vanquish™ Neo UHPLC System, 300 nL / min, the HPLC column is Acclaim™ PepMap™ RSLC C18 2pim 50 cmx75 pm, and about 1 pig of proteins used to prepare the samples for proteomics LC / MS analysis, optionally using an overall HPLC gradient time of 180 min. In another embodiment, the mass spectrometer is Orbitrap Fusion™ Lumos™ Tribrid™, the HPLC system is Vanquish™ Neo UHPLC System, 300 nL / min, the HPLC column is Acclaim™ PepMap™ RSLC C18 2pim 50 cmx75 pm, and about 2.5 pig of proteins used to prepare the samples for proteomics LC / MS analysis, optionally using an overall HPLC gradient time of 180 min.

[0305] Identification of the proteins is carried out using a software such as, for example, Mascot v2.8.2 and Scaffold v5.0.0. Database for protein identification include, for example, SwissProt Human (20221118, 20607 entries). The criteria of protein identifications is a peptide false discovery rate (FDR) of 1 % (1 or 2 peptide(s) minimum). FDR relates to the probability to correctly identify a peptide / protein. Thus, a FDR of 1% indicates that from a list of 100 identified proteins, 1 of these proteins may be misidentified.

[0306] Exemplary LC / MS detection conditions are provided in the table below: In some embodiments, one or more of Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) is not detected in the exosomes of the present invention by LC / MS analysis as described herein. In some embodiments, Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected in the exosomes of the present invention by LC / MS analysis as described herein. In some embodiments, one or more of C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1, cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) is not detected in the exosomes of the present invention by LC / MS analysis as described herein. In some embodiments, C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1, cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are not detected in the exosomes of the present invention by LC / MS analysis as described herein.

[0307] In some embodiments, Hypoxia up-regulated protein 1 (HYOU1) is not detected in the exosomes of the present invention by LC / MS analysis as described herein. In some embodiments, Apolipoprotein A-IV (APOA4) is not detected in the exosomes of the present invention by LC / MS analysis as described herein.

[0308] In some embodiments, Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) is not detected in the exosomes of the present invention by LC / MS analysis as described herein.

[0309] The term "not detected” as used herein include proteins or peptides not present in a sample, or present in an amount too low to be detected by LC / MS analysis, for example less than fmol concentration. In some embodiments, reference to proteins and peptides not detected refers to proteins and peptides sequences not detected by the LC / MS analysis according to any of the embodiments for detection by LC / MS described above.

[0310] However, importantly, the exosomes described herein were found to comprise proteins known in the literature to be present in exosomes derived from natural human breast milk that are key to infant health and development. These proteins include Lactotransferrin (LTF), Annexin A2 (ANXA2) and Lactadherin (MFGE8). In some embodiments, these proteins include Lactotransferrin (LTF), Annexin A2 (ANXA2) and Lactadherin (MFGE8), MUC1 and tetraspanins CD9, CD81 and CD63.

[0311] Thus, a human breast milk exosome described herein may not comprise one or more proteins selected from C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4), Procollagen-lysine,2- oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0312] In some embodiments, the exosome does not comprise C4a anaphylatoxin (CFA).

[0313] In some embodiments, the exosome does not comprise Hypoxia up-regulated protein 1 (HYOU1).

[0314] In some embodiments, the exosome does not comprise Apolipoprotein A-IV (APOA4).

[0315] In some embodiments, the exosome does not comprise Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1).

[0316] In some embodiments, the exosome does not comprise Threonine— tRNA ligase 1 cytoplasmic (TARS1).

[0317] In some embodiments, the exosome does not comprise Cytosolic non-specific dipeptidase (CNDP2).

[0318] In some embodiments, the exosome does not comprise one or more of Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1).

[0319] In some embodiments, the exosome does not comprise proteins C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0320] In some embodiments, the exosome comprises one or more of the proteins listed in Table 1. In some embodiments, the exosome comprises all of the proteins listed in Table 1 .

[0321] Table 1

[0322] | Aminopeptidase N | ANPEP |

[0323] In some embodiments, the exosome comprises Lactotransferrin (LTF), Annexin A2 (ANXA2) and / or Lactadherin (MFGE8). In some embodiments, the exosome comprises Lactotransferrin (LTF), Annexin A2 (ANXA2) and Lactadherin (MFGE8). In some embodiments, the exosome comprises Lactotransferrin (LTF), Annexin A2 (ANXA2) and Lactadherin (MFGE8), MUC1 and tetraspanins CD9, CD81 and CD63.

[0324] In some embodiments, the exosome comprises said one or more proteins described anywhere herein in substantially the same concentration as the same one or more proteins in exosomes derived from natural human breast milk.

[0325] In some embodiments, the exosome comprises said one or more proteins described anywhere herein in different concentrations as the same one or more proteins in exosomes derived from natural human breast milk. In some embodiments, said one or more proteins may be present in a higher concentration compared to the concentration of said one or more proteins in exosomes derived from natural human breast milk. In some embodiments, said one or more proteins may be present in a lower concentration compared to the concentration of said one or more proteins in exosomes derived from natural human breast milk.

[0326] A composition comprising the exosomes or population of exosomes as described anywhere herein can be for use in promoting or supporting brain development. In some further embodiments, the composition can comprise I) the exosomes or population of exosomes as described anywhere herein and ii) one or more proteins that are absent from the exosomes of the present invention but are present in exosomes derived from natural human breast milk. Said proteins may be selected from but not limited to one or more of proteins C4a anaphylatoxin (CFA), Hypoxia up- regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1, cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0327] In some embodiments, the composition is a nutritional composition. Accordingly, in some embodiments, the present invention relates to a nutritional composition comprising I) the exosomes or population of exosomes as described anywhere herein and ii) one or more proteins that are absent from the exosomes of the present invention but are present in exosomes derived from natural human breast milk. Said proteins may be selected from but not limited to one or more of proteins Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagenlysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1). In another embodiment, said proteins may be selected from but not limited to one or more of proteins C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1, cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0328] In some embodiments, the composition is a pharmaceutical composition. Accordingly, in some embodiments, the present invention relates to a pharmaceutical composition comprising I) the exosome or population of exosomes as described anywhere herein, ii) a pharmaceutically acceptable excipient and ill) one or more proteins that are absent from the exosomes of the present invention but are present in exosomes derived from natural human breast milk. Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine, 2-oxoglutarate 5- dioxygenase 1 (PLOD1). In another embodiment, said proteins may be selected from but not limited to one or more of proteins C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PL0D1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0329] Exosome miRNA profile miRNA analysis of the exosomes described herein surprisingly demonstrated that the exosomes possess substantially the same miRNA profile to that of exosomes derived from natural human breast milk.

[0330] In particular, exosomes described herein were surprisingly found to comprise miRNAs known in the literature to be present in exosomes derived from natural human breast milk that are key to infant health and development. These miRNAs include miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

[0331] Thus, in some embodiments, a human breast milk exosome described herein comprises one or more miRNAs selected from miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

[0332] In some embodiments, the exosome comprises miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

[0333] In some embodiments, the exosome comprises said one or more miRNAs described anywhere herein in substantially the same concentration as the same one or more miRNAs in exosomes derived from natural human breast milk.

[0334] In some embodiments, the exosome comprises said one or more miRNAs described anywhere herein in different concentrations as the same one or more miRNAs in exosomes derived from natural human breast milk. In some embodiments, said one or more miRNAs may be present in a higher concentration compared to the concentration of said one or more miRNAs in exosomes derived from natural human breast milk. In some embodiments, said one or more miRNAs may be present in a lower concentration compared to the concentration of said one or more miRNAs in exosomes derived from natural human breast milk.

[0335] Exosome lipidomics

[0336] Lipid analysis of the exosomes described herein surprisingly demonstrated that the exosomes possess substantially the same lipid profile to that of exosomes derived from natural human breast milk.

[0337] In particular, exosomes described herein were surprisingly found to comprise lipid classes known in the literature to be present in exosomes derived from natural human breast milk that are key to infant health and development. These lipids include cholesterol esters, ceramides, triacylglycerides, diacylglycerides, lyso-phospholipids, total phospholipids, alkanyl phospholipids, and alkenyl phospholipids.

[0338] Of these key lipid classes, phospholipids in particular are considered to be key for exosome structure and function. Exosomes of the present invention were also surprisingly found to comprise phospholipid sub-classes known in the literature to be present in exosomes derived from natural human breast milk. These phospholipids include sphingomyelins, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0339] Thus, in some embodiments, a human breast milk exosome described herein can comprise one or more lipids selected from cholesterol esters, ceramides, triacylglycerides, diacylglycerides, total phospholipids, alkanyl phospholipids, and alkenyl phospholipids.

[0340] In some embodiments, the exosome comprises cholesterol esters, ceramides, triacylglycerides, diacylglycerides, total phospholipids, alkanyl phospholipids, and alkenyl phospholipids. In some embodiments, the exosome comprises one or more phospholipids selected from sphingomyelins, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0341] In some embodiments, the exosome comprises sphingomyelins, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0342] In some embodiments, the exosome comprises said one or more lipids described anywhere herein in substantially the same concentration as the same one or more lipids in exosomes derived from natural human breast milk.

[0343] In some embodiments, the exosome comprises said one or more lipids described anywhere herein in different concentrations as the same one or more lipids in exosomes derived from natural human breast milk. In some embodiments, said one or more lipids may be present in a higher concentration compared to the concentration of said one or more lipids in exosomes derived from natural human breast milk. In some embodiments, said one or more lipids may be present in a lower concentration compared to the concentration of said one or more lipids in exosomes derived from natural human breast milk.

[0344] Exosome size

[0345] Size analysis of the exosomes described herein demonstrated that the exosomes are substantially the same size in diameter as exosomes derived from natural human breast milk.

[0346] Thus, in some embodiments, a human breast milk exosome described herein has a diameter size of 50 to 100nm, preferably 60-80nm, more preferably 65 to 75nm.

[0347] In some embodiments, the exosome has a diameter size of 65 to 75nm.

[0348] It will be understood that any features of the exosomes described anywhere herein may be combined. For example, features relating to the proteome of the exosome may be combine with features relating to the miRNA profile, lipid profile and / or size profile.

[0349] Additional Embodiments of the Invention

[0350] The present invention may be described according to the following numbered statements:

[0351] 51. A method of promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a subject comprising administering an isolated human breast milk exosome, wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2- oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome, to a subject in need thereof, optionally wherein the subject has sub-optimal brain development, further optionally is born pre-term, is small for gestational age, is low-birth weight (LBW) or has experienced intra-uterine growth retardation (IUGR) or has suffered from growth stunting because of malnutrition, such as suboptimal intrauterine nutrition and / or disease.

[0352] 52. A method of treating a disease or disorder selected from a demyelinating disease, a neurodevelopmental disorder, a neurocognitive disorder, or a neuropsychiatric disorder in a subject in need thereof comprising administering to the subject an isolated human breast milk exosome, wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome. 53. The method of statement 2, wherein the disease or disorder is a demyelinating disease, optionally selected from: multiple sclerosis, neuromyelitis optica spectrum disorder, transverse myelitis, acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

[0353] 54. The method of statement 2, wherein the disease or disorder is a neurodevelopmental disorder, optionally selected from: attention-defici t / hy peractivity disorder, autism spectrum disorders, communication disorders, intellectual disability, motor disorders, and specific learning disorder.

[0354] 55. The method of statement 2, wherein the disease or disorder is a neurocognitive disorder, optionally selected from: Alzheimer's disease, frontotemporal lobar degeneration, Lewy body disease, vascular disease, traumatic brain injury, substance or medication use, HIV infection, prion disease, Parkinson's disease, and Huntington's disease.

[0355] 56. The method of statement 2, wherein the disease or disorder is a neuropsychiatric disorder, optionally selected from: seizure, attention deficit disorder, cognitive deficit disorder, palsies, uncontrolled anger, migraine headaches, addictions, eating disorders, depression, and anxiety.

[0356] 57. Use of an isolated human breast milk exosome, wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome, for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a healthy subject.S8.

[0357] The method of any one of statements 1 to 6 or the use of statement 7, wherein C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4), Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are not detected by liquid chromatography - mass spectrometry in the exosome.

[0358] S9. A method of promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a subject comprising administering an isolated human breast milk exosome, wherein the exosome does not comprise one or more proteins selected from C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4), Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2), to a subject in need thereof, optionally wherein the subject has sub-optimal brain development, further optionally is born pre-term, is small for gestational age, is low-birth weight (LBW) or has experienced intra-uterine growth retardation (IUGR) or has suffered from growth stunting because of malnutrition, such as suboptimal intrauterine nutrition and / or disease.

[0359] 510. A method of treating a disease or disorder selected from a demyelinating disease, a neurodevelopmental disorder, a neurocognitive disorder, or a neuropsychiatric disorder in a subject in need thereof comprising administering to the subject an isolated human breast milk exosome, wherein the exosome does not comprise one or more proteins selected from C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2).

[0360] 511. The method of statement 10, wherein the disease or disorder is a demyelinating disease, optionally selected from: multiple sclerosis, neuromyelitis optica spectrum disorder, transverse myelitis, acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy, and central pontine myelinolysis. 512. The method of statement 10, wherein the disease or disorder is a neurodevelopmental disorder, optionally selected from: attention-defici t / hy peractivity disorder, autism spectrum disorders, communication disorders, intellectual disability, motor disorders, and specific learning disorder.

[0361] 513. The method of statement 10, wherein the disease or disorder is a neurocognitive disorder, optionally selected from: Alzheimer's disease, frontotemporal lobar degeneration, Lewy body disease, vascular disease, traumatic brain injury, substance or medication use, HIV infection, prion disease, Parkinson's disease, and Huntington's disease.

[0362] 514. The method of statement 10, wherein the disease or disorder is a neuropsychiatric disorder, optionally selected from: seizure, attention deficit disorder, cognitive deficit disorder, palsies, uncontrolled anger, migraine headaches, addictions, eating disorders, depression, and anxiety.

[0363] 515. Use of an isolated human breast milk exosome, wherein the exosome does not comprise one or more proteins selected from C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2), for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a healthy subject.

[0364] 516. The method of any one of statements 1 to 6, or 8 to 14 or the use of any one of statements 7, 8 , or 15, wherein the exosome comprises lactotransferrin (LTF), annexin A2 (ANXA2) and lactadherin (MFGE8), MUC1 and tetraspanins CD9, CD81 and CD63.

[0365] 517. The method of any one of statements 1 to 6, 8 to 14, or 16 or the use of any one of statements 7, 8, 15, or 16, wherein the exosome comprises one or more miRNAs selected from miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

[0366] 518. The method of any one of statements 1 to 6, 8 to 14, 16, or 17 or the use of any one of statements 7, 8, or 15 to 17, wherein the exosome comprises one or more lipids selected from cholesterol esters, ceramides, triacylglycerides, diacylglycerides, lyso-phospholipids, total phospholipids, alkanyl phospholipids, and alkenyl phospholipids.

[0367] 519. The method of any one of statements 1 to 6, 8 to 14, or 16 to 18 or the use of any one of statements 7, 8, or 15 to 18, wherein the exosome comprises one or more phospholipids selected from sphingomyelins, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

[0368] 520. The method of any one of statements 1 to 6, 8 to 14, or 16 to 19or the use of any one of statements 7, 8, or 15 to 19, wherein the exosome has a diameter of 50 to 100nm.

[0369] 521 . The method of statement 20 or the use of statement 20, wherein the exosome has a diameter size of 65 to 75nm.

[0370] 522. The method of any one of statements 1 to 6, 8 to 14, or 16 to 21 or the use of any one of statements 7, 8, or 15 to 21, wherein the exosome has the same function as exosomes derived from natural human breast milk.

[0371] 523. The method of any one of statements 1 to 6, 8 to 14, or 16 to 22 or the use of any one of statements 7, 8, or 15 to 22, wherein the exosome has the same structure as exosomes derived from natural human breast milk.

[0372] 524. The method of any one of statements 1 to 6, 8 to 14, or 16 to 23 or the use of any one of statements 7, 8, or 15 to 23, wherein the exosome is comprised in a population of exosomes. 525. The method of any one of statements 1 to 6, 8 to 14, or 16 to 24 or the use of any one of statements 7, 8, or 15 to 24, wherein the exosome product is obtainable according to an in vitro method, the method comprising:

[0373] A) Generating lactocyte mammary-like gland organoids derived from mammalian induced pluripotent stem cells (miPSC)

[0374] B) Secreting a mammalian milk like product from said lactocytes, and

[0375] C) Purifying the exosomes from the mammalian milk like product to remove impurities, optionally by chromatography or filtration or ultracentrifugation, in order to isolate the exosomes, wherein step A) comprises culturing the miPSCs in a culture medium comprising BMP4 and / or RA

[0376] 526. The method or the use of statement 25, wherein step A) comprises:

[0377] I) culturing the mammalian induced pluripotent stem cells (miPSCs) in a culture medium comprising BMP4 and / or RA to generate embryoid bodies (EBs), and ii) growing the EBs to generate a population of mammary cells.

[0378] 527. The method or the use of statement 26, wherein the culturing step I) comprises culturing the miPSCs in MammoCult medium and BMP4, in a 3D-suspension culture system, for example 3D-suspension condition, thereby directing the IPSCs to differentiate towards non-neural ectoderm cells, optionally for at least 12 days.

[0379] 528. The method or the use of statement 26, wherein the culturing step I) comprises culturing the miPSCs in MammoCult medium and BMP4, in a 3D-suspension culture system, for example 3D-suspension condition, thereby directing the IPSCs to differentiate towards non-neural ectoderm cells, optionally for no more than 8 days.

[0380] 529. The method or the use of statements 26 or 27, wherein the growing step II) comprises growing the formed EBs in a 3D embedding system comprising RA, for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen I for at least 30 days, for example for 32 days, to generate lactocytes.

[0381] 530. The method or the use of statements 26 or 27, wherein the growing step II) comprises growing the formed EBs in a 3D embedding system comprising RA, for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen I for no more than 25 days, to generate lactocytes.

[0382] 531 . The method or the use of statement 29, wherein step II) is distinguished into further substeps and comprises the following steps: II) and ill):

[0383] II) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult proliferation supplement, Parathyroid hormone (pTHrP), and RA for 5 days, ill) promotion of branch and alveolar differentiation and mammary cell specification by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGFIO and HGF for 20 days.

[0384] 532. The method or the use of statement 30, wherein step II) is distinguished into further substeps and comprises the following steps: II) and ill):

[0385] II) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I floated in EpiCultB medium supplemented with EpiCult proliferation supplement, Parathyroid hormone (pTHrP), and RA for 5 days, ill) promotion of branch and alveolar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGFIO and HGF for 15 days. 533. The method or the use of any one of statements 25 to 32, wherein the mammary gland cells are human mammary gland cells.

[0386] 534. The method or the use of any one of statements 25 to 33, wherein BMP4 is added to the culture medium between day 0 and day 10, preferably between day 0 and day 3, where day 0 is the time point where the iPSCs are first added to the culture medium.

[0387] 535. The method or the use of any one of statements 25 to 34, wherein BMP4 is added to the culture medium for 3 days.

[0388] 536. The method or the use of any one of statements 25 to 35, wherein RA is added to the culture medium between day 10 and day 15, optionally between day 6 and day 11 , where day 0 is the time point where the IPSCs are first added to the culture medium.

[0389] 537. The method or the use of any one of statements 25 to 36, wherein RA is added to the culture medium for 5 days.

[0390] 538. The method or the use of any one of statements 25 to 37, wherein BMP4 is added to the culture medium in a concentration of 5 to 20 ng / mL, preferably 5 ng / mL, 10 ng / mL or 20 ng / mL.

[0391] 539. The method or the use of any one of statements 25 to 38, wherein RA is added to the culture medium in a concentration of 1 piM.

[0392] 540. The method or the use of any one of statements 25 to 39, wherein the EBs express one or more mammary gland positive progenitor-cell markers, optionally selected from EpCAM, CD49f, MUC1 and GAT A3.

[0393] 541 . The method or the use of any one of statements 25 to 40, wherein the EBs have increased expression of one or more mammary gland positive progenitor-cell markers compared to the expression level of said mammary gland positive progenitor-cell markers in EBs not treated with BMP4 and RA.

[0394] 542. The method or the use of any one of statements 25 to 41, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the EBs express one or more mammary gland positive progenitor-cell markers, optionally wherein at least 35% of EBs express EpCAM and CD49f mammary gland positive progenitor-cell markers, optionally wherein at least 15% of EBs express MUC1 and EpCAM mammary gland positive progenitor-cell markers, and / or optionally wherein at least 20% of EBs express EpCAM and GAT A3 mammary gland positive progenitor-cell markers.

[0395] 543. The method or the use of any one of statements 25 to 42, wherein the EBs express one or more milk-specific bioactive markers, optionally osteopontin (OPN).

[0396] 544. The method or the use of any one of statements 25 to 43, wherein the EBs have increased expression one or more milk-specific bioactive markers, optionally increased expression of osteopontin (OPN).

[0397] 545. The method or the use of any one of statements 25 to 44, wherein the EBs have increased secretion one or more milk-specific bioactive markers, optionally increased secretion of osteopontin (OPN).

[0398] 546. The method or the use of any one of statements 25 to 45, wherein step A) is between a 30-day and 45-day process, optionally less than a 45-day process, optionally less than a 44-day process, optionally less than a 35-day process, optionally less than a 31 -day process.

[0399] 547. The method or the use of any one of statements 25 to 46, wherein the method is for producing a human milk like product, wherein step A) further comprises: i) directing hiPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium comprising BMP4, for example MammoCult medium and BMP4, in an appropriate 3D culture system, for example 3D-suspension condition, for at least 12 days and

[0400] II) growing the formed mEBs (mammospheres) in an appropriate 3D embedding system comprising RA, for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen I and RA for at least 30 days, for example for 32 days, to generate lactocytes.

[0401] 548. The method or the use of any one of statements 25 to 47, wherein the method is for producing a human milk substitute product, wherein step A) further comprises: i) directing hiPSCs to differentiate towards non-neural ectoderm cells by culturing them in an appropriate culture medium comprising BMP4, for example MammoCult medium and BMP4, in an appropriate 3D culture system, for example 3D-suspension condition, for no more than 8 days and

[0402] II) growing the formed mEBs (mammospheres) in an appropriate 3D embedding system comprising RA, for example a mixed floating gel composed of matrix protein such as Matrigel and / or Collagen I and RA for no more than 25 days, to generate lactocytes.

[0403] 549. The method or the use according to statement 47 wherein step A)i) is defined as follows:

[0404] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL or in medium mTeSR™ for two days, and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in complete MammoCult medium comprising the basal medium, proliferation supplement and supplemented with BMP4, heparin, and hydrocortisone for 10 days, and wherein step A)ii) is distinguished into further substeps and comprises the following steps: ii), ill) and iv): ii) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) and RA for 5 days, ill) promotion of branch and alveolar differentiation and mammary cell specification by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 20 days, and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 7 days.

[0405] 550. The method or the use according to statement 48 wherein step A)i) is defined as follows:

[0406] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL or in medium mTeSR™ for two days, and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in complete MammoCult medium comprising the basal medium, proliferation supplement and supplemented with BMP4, heparin, and hydrocortisone for 6 days, and wherein step A)ii) is distinguished into further substeps and comprises the following steps: ii), ill) and iv): ii) incubation of mEBs (mammospheres) in complete EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) and RA for 5 days, iii) promotion of branch and alveolar differentiation and mammary cell specification by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 15 days, and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 5 days.

[0407] 551 . The method or the use according to statement 47 wherein step A)i) is defined as follows:

[0408] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL for two days, and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin and BMP4 for 10 days, and wherein step A)ii) is distinguished into further substeps and comprises the following steps ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I floated in EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) and RA for 5 days, iii) promotion of branch and alveolar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 20 days, and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 7 days.

[0409] 552. The method or the use according to statement 48 wherein step A)i) is defined as follows:

[0410] I) generation of embryoid bodies (EBs) from hiPSCs by incubation in standard IPSC medium E8 comprising DMEM / F12, L-ascorbic acid-2-phosphate magnesium, sodium selenium, FGF2, insulin, NaHCOa and transferrin, TGFpl or NODAL for two days, and producing mEBs (mammospheres) highly enriched in non-neural ectodermal cells by incubation of EBs in MammoCultB medium supplemented with MammoCult proliferation supplement, hydrocortisone, heparin and BMP4 for 6 days, and wherein step A)ii) is distinguished into further substeps and comprises the following steps ii), iii) and iv): ii) embedding the formed mEBs (mammospheres) in a mixture of Matrigel and Collagen I floated in EpiCultB medium supplemented with EpiCult proliferation supplement and Parathyroid hormone (pTHrP) and RA for 5 days, iii) promotion of branch and alveolar differentiation and mammary cell specification by incubating embedded mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FGF10 and HGF for 15 days, and iv) induction of milk protein expression by incubating mEBs (mammospheres) in EpiCultB medium supplemented with EpiCult proliferation supplement, hydrocortisone, insulin, FBS, prolactin, progesterone and p-estradiol for 5 days.

[0411] 553. The method or the use of any one of statements 25 to 52, wherein Step A) is conducted in 3D suspension culture conditions.

[0412] 554. The method or the use of any one of statements 25 to 53, wherein step C) further comprises formulating the isolated exosomes into a powder form, optionally by spray drying or freeze drying, or liquid form. 555. The method or the use of any one of statements 25 to 54, wherein step C) further comprises sterilising the isolated exosomes.

[0413] 556. The method or the use of any one of statements 25 to 55, wherein step C) further comprises storing the isolated exosomes at below freezing, optionally at -80°C.

[0414] 557. The method or the use of any one of statements 25 to 56, further comprising formulating the isolated exosomes with supplementary nutritional ingredients.

[0415] 558. The method or the use of any one of statements 25 to 57, wherein the isolated exosomes are dispensed into a container for consumption.

[0416] 559. The method or the use according to any one of statements 25 to 58, wherein the exosome is a human breast milk exosome.

[0417] It should be appreciated that the various aspects and embodiments of the detailed description as disclosed herein are illustrative of the specific ways to make and use the invention and do not limit the scope of invention when taken into consideration with the claims and the detailed description. It will also be appreciated that features from aspects and embodiments of the invention may be combined with further features from the same or different aspects and embodiments of the invention.

[0418] As used in this detailed description and the appended claims, the singular forms "a,” "an” and "the” include plural referents unless the context clearly dictates otherwise.

[0419] Figures

[0420] Fig. 1 : shows the differentiation of human induced pluripotent stem cells (hiPSCs) according to the protocol outlined in Ying Qu and as applied in one alternative in step A) of the inventive methods.

[0421] Fig. 2: shows the differentiation of human induced pluripotent stem cells (hiPSCs) according to step A).

[0422] Fig. 3: shows the differentiation of human induced pluripotent stem cells (hiPSCs) according to the preferred and particularly preferred embodiments for step A) of the inventive methods.

[0423] Fig. 4: shows that three-dimensional organotypic cultures of hiPSCs as produced according to the methods of Fig. 2 are highly permissive for mammary glands specification. mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8 and CSN2 for 3D-differentiation (42 days) protocol are shown. Markers from left to right: Stages of Pluripotency (Nanog), Lineage (ectoderm & endoderm) (TUBB3, FOXA2), Basal- cell / myoepithelial markers (TP63, KR-14), Luminal epithelial markers (EpCAM, KRT8), and milk proteins (CSN2 (Casein Beta)).

[0424] Fig. 5: shows the two-dimensional organotypic culture of hiPSCs produced as a comparative example. mRNA expression of Nanog, TUBB3, FOXA2, TP63, KR-14, EpCAM, KRT8 and CSN2 for 2D-differentiation (31 days) protocols are shown. Markers from left to right: Sages of Pluripotency (Nanog), Lineage (ectoderm & endoderm) (TUBB3, FOXA2), Basal-cell / myoepithelial markers (TP63, KR-14), Luminal epithelial markers (EpCAM, KRT8), and milk proteins (CSN2 (Casein Beta)).

[0425] Fig. 6: shows a schematic illustrations of different mammary-gland differentiation protocols. Scheme summarizing different procedures for the generation of mammary gland progenitors in 42 days (a), using bone morphogenetic protein 4 (BMP4) (b), retinoic acid (RA) (c), combinations of BMP4 and RA (d) and their combination in shortened time-course (31 days) (e). The red boxes indicate the period of addition of BMP4 and / or RA.

[0426] Fig 7: shows combinatorial effects of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) on mammarygland differentiation of induced pluripotent stem cells (IPSCs) using a 3D-organoid model, a-c, Flow cytometry quantification of the number of mammary gland positive progenitor-cells using several identified markers: EpCAM, CD49f, MUC1 and GAT A3 during the differentiation time-course (day 25 (for control protocols) and day 20 (for shortened time-course protocol)) and pre-induction stages (day 35 (for control protocols including BMP4 and RA), and day 26 (for shortened time-course protocol)) and d-f, post-induction period (days 42 (for control protocols including BMP4 and RA) and 31 (for shortened time-course protocol)). * describes shoriened-modified protocols (31 days) including BMP4 and RA combinations.

[0427] Fig 8: shows expression of different mammary epithelium markers in lactocytes derived from IPSCs in normal length and shortened protocols (a-h).

[0428] Fig 9: shows Liquid chromatography-mass spectrometry (LC-MS / MS) based targeted proteomic analysis of osteopontin protein secretion. Osteopontin protein secretion was detected in the media of cultures of IPS- derived mammary-gland cells using control differentiation method, or with bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and in the combination of the two factors.

[0429] Fig 10: a, Venn diagram shows the number of proteins identified in EVs purified from conditioned media (Day 39-41) only (circle on the left hand side), in EVs purified from human milk only (circle on the right hand side), and in both types of EVs (overlapping circles); b, Table shows the list of the top 50 proteins (based on FDR) detected in both types of EVs (human milk and conditioned media).

[0430] Fig 11 : a, Venn diagram shows the number of miRNAs significantly downregulated in EVs purified from conditioned media (Day 39-41) compared to human milk EVs (circle), significantly upregulated in conditioned media EVs compared to human milk EVs (square), and not differentially expressed in both types of EVs (overlapping circle and square shapes), b, Venn diagram shows result obtained from subsequent repetition of the experiment of Example 6 with 2 additional batches of cell-based EVs. Venn diagram shows the number of miRNAs significantly downregulated in EVs purified from conditioned media (Day 39-41) compared to human milk EVs (circle), significantly upregulated in conditioned media EVs compared to human milk EVs (square), and not differentially expressed in both types of EVs (overlapping circle and square shapes), c, Table shows the list of the top 4 miRNAs (based on FDR) expressed in both types of EVs (human milk and conditioned media).

[0431] Fig 12: shows the proportions of lipid (left panel) and phospholipid (right panel) classes found in EVs purified from human milk and conditioned media (Day 39-41). Data is expressed a percentage of total lipid and phospholipid, respectively. TG: Triacylglycerides; DG: Diacylglycerides; PL: Total phospholipids; Lyso: Lyso-phospholipid; Alkanyl: Phospholipid-O; Alkenyl: Phospholipid-P; CE: Cholesterol esters; CER: Ceramides; SM: Sphingomyelins; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PI: Phosphatidylinositol; PS: Phosphatidylserine. The specific lipids and phospholipids in the bar charts, starting from the bottom to the top of the charts, correspond to the lipids or phospholipids in the abbreviated lists, starting left to right. Fig 13: shows the median particle size (nm) of EVs purified from conditioned media (Day 41) and human milk, analysed my Nano Flow Cytometry.

[0432] Fig. 14: shows Lacto EVs (green) present within rat cortical astrocytes that have been stained with GFAP (red). The arrows indicate the presence of EVs in both the cytoplasm and astrocytic processes.

[0433] Fig. 15: shows Lacto EVs (green) present within rat cortical neurons that have been stained with Beta III tubulin (red). Nuclei are labeled using Dapi (blue).

[0434] Fig 16: shows neuronal treatment with milk and Lacto exosomes.

[0435] Fig 17: shows the treatment of neurons with astrocytic conditioned media after treatment with milk and Lacto exosomes.

[0436] Fige 18: shows the preparation of the astrocytic conditioned media.

[0437] Fig 19: shows developmental stages grouped in three DIV (day in vitro) intervals: Early development (DIV 8-11), Intermediate development (DIV 12-15), and Late development (DIV 16-19).

[0438] Fig. 20: shows data for neurons treated with conditioned media from astrocytes treated with Human Milk EVs.

[0439] Fig. 21 : A) and B) show data for neurons treated with conditioned media from astrocytes treated with lactocyte EVs.

[0440] C) and D) show replicate data pooled with the data from A) and B).

[0441] Fig. 22: shows data for direct neuronal treatment with Human Milk EVs.

[0442] Fig. 23: A) and B) show data for direct neuronal treatment with lactocyte EVs. C) and D) show replicate data pooled with the data from A) and B).

[0443] Fig 24: shows an illustration of the experimental protocol.

[0444] Fig 25: shows a barplot of selected regulated pathways for cortex NMS HM (Human Milk) EVs vs NMS PBS.

[0445] Fig 26: shows a barplot of selected regulated pathways for cortex NMS Lacto (lactocyte) EVs vs NMS PBS.

[0446] Fig 27: shows a barplot of selected regulated pathways for hippocampus NMS HM EVs vs NMS PBS.

[0447] Fig 28: shows a barplot of selected regulated pathways for hippocampus NMS Lacto EVs vs NMS PBS.

[0448] Fig 29: shows a barplot of selected regulated pathways for hippocampus NMS HM EVs vs NMS PBS.

[0449] Fig 30: shows a barplot of selected regulated pathways for hippocampus NMS Lacto EVs vs NMS PBS.

[0450] Fig 31 : shows a barplot of selected regulated pathways for cerebellum NMS HM EVs vs NMS PBS.

[0451] Fig 32: shows a barplot of selected regulated pathways for cerebellum NMS Lacto EVs vs NMS PBS.

[0452] Experimental section

[0453] Example 1

[0454] Cultivation and differentiation of hiPSCs into lactocytes to obtain a human milk like product

[0455] Lactocytes are cultured starting from ihPSCs according to the procedure described in Ying Qu et al, Stem Cell Report vol 8, 205-215 February 14th2017 and the human milk like product thereby secreted is collected and can be used in therapy and / or as a breastfeeding substitute according to the present invention.

[0456] Example 2

[0457] Cultivation and differentiation of hiPSCs into 3D-lactocytes to obtain a human milk like product Lactocytes are cultured starting from hiPSCs according to the method of the present invention following steps A) and B) as described above) and the human milk like product thereby secreted is collected and can be used in therapy and / or as a breastfeeding substitute according to the present invention.

[0458] Example 3

[0459] Alternative methods of cultivation and differentiation of hiPSCs into lactocytes to obtain a human milk like product

[0460] Efficient lactocytes differentiation from hiPSCs can be obtained from alternative culture conditions including conditions 1 to 4 as below described:

[0461] 1 . 2D culture on vitronectin coated plates as monolayer of cells derived from the EBs and cultured for at least 28 days in a medium containing (RPM1 1640 with L-glutamine; Fetal bovine serum (FBS); Insulin; Epidermal growth factor (EGF); hydrocortisone; Pen-Strep (penicillin / streptomycin : antibiotic-antimycotic solution).

[0462] 2. 2D culture on vitronectin coated plates of attached aggregates (EBs) of cells derived from the EBs and cultured for at least 28 days in a medium containing (RPM1 1640 with L-glutamine; Fetal bovine serum (FBS); Insulin; Epidermal growth factor (EGF); hydrocortisone; Pen-Strep (antibiotic-antimycotic solution).

[0463] 3. 3D culture in suspension in MammoCult medium for at least 10 days and then culture in mixed floating gels (for example Matrigel and Collagen 1) for another 5 days in a specific medium (for example EpiCultB) in presence of Parathyroid hormone followed by 25 days in presence of insulin, HGF, hydrocortisone and FGF10;

[0464] 4. 3D culture of EBs in suspension (ultra low adherent plate) in MammoCult medium for at least 10 days and then in suspension culture for another 5 days in a specific medium (for example EpiCultB) in presence of Parathyroid hormone followed by 25 days in presence of insulin, HGF, hydrocortisone and FGF10.

[0465] Example 4

[0466] 2D- and 3D-lactocyte differentiation based on human-induced pluripotent stem cell (hiPSC) line 603

[0467] (a) 3D-lactocyte differentiation based on human-induced pluripotent stem cell (hiPSC) line 603:

[0468] The human-induced pluripotent stem cell (hiPSC) line 603 was used for 3D-lactocyte differentiation. The human-induced pluripotent stem cell (hiPSC) line 603was purchased from Fujifilm Cellular Dynamics, Inc (FCDI).

[0469] (i) For the 3D differentiation protocol (according to the invention), EBs (spheroids) were formed by incubating single cells of hiPSC in E8 medium with 10uM rock inhibitor at 37°C, 5% CO2 in rotation at 95 rpm overnight.

[0470] Second day, medium was replaced with E8 (day -2-day 0).

[0471] Next day, medium was replaced with Mammol medium (MammoCult - medium with proliferation supplements, heparin (4pig / mL), and hydrocortisone (0.48pig / mL) with penicillin / streptomycin) for 10 days (day 0-day 10). Medium was changed every second day.

[0472] (ii) The differentiation was followed by 5 days in Mammo2 medium (EpiCultB + supplements, pTHrP 10Ong / ml plus penicillin / streptomycin). Culture medium was changed every 3 days (day 10-day 15). (iii) In order to induce branching epithelial structure, alveolar differentiation and mammary cell specification, mEBs (spheroids / mammospheres) were fed with Mammo3 medium (complete EpiCultB, hydrocortisone (1 pig / ml), insulin (10 pig / ml), FGF10 (50 ng / ml), HGF (50 ng / ml) and penicillin / streptomycin) for 20 days. Medium was changed every 3 days (day 15-day 35).

[0473] (iv) Finally, to induce the milk bioactive production (3D), we used the Mammo4 medium (complete EpiCultB, 10% FBS, prolactin (10 pig / ml), hydrocortisone (1 pig / ml), insulin (10 pig / ml), progesterone, p-estradiol and penicillin / streptomycin for 7 days and medium was changed every 3 days (day 35-day 42). During all the differentiation procedure, spheroids were maintained in the suspension culture (rotating at 95 rpm). The differentiation procedure ended at day 42. Results are displayed in Figure 4.

[0474] (b) 2D-lactocyte differentiation based on human-induced pluripotent stem cell (hiPSC) line 603

[0475] The human-induced pluripotent stem cell (hiPSC) line 603 was used also for 2D-lactocyte differentiation. The human-induced pluripotent stem cell (hiPSC) line 603was purchased from Fujifilm Cellular Dynamics, Inc (FCDI).

[0476] For the 2D-differentiation protocol (used for comparison), we used the Lacto medium during all the differentiation stages (RPM1 1640, 20% FBS, 1 mM glutamine, 4 pig / ml insulin, 20 ng / ml EGF, 0.5pig / ml hydrocortisone with penicillin / streptomycin). Cells were incubated at 37°C, 5% CO2. Medium was replaced every second day. Results are displayed in Figure 5.

[0477] (c) Results

[0478] The different differentiation stages during lactocyte derivation were captured using quantitative RT-PCR (Figure 4, 3D-differentiation, Figure 5, 2D-differentiation). In both 2D- and 3D-settings, NaNog expression as a marker for pluripotency is decreased while cells are passing towards the maturation and differentiation. The neuroectodermal and endodermal markers, TUBB3 (Tubulin Beta 3 Class III) and Forkhead box protein A2 (FOXA2) were not expressed significantly in 3D-format and TUBB3 elevation is only captured in 2D-setting. This demonstrates that hiPSCs are patterned towards the non-neural ectodermal lineage, thus enriching mammary progenitors in 3D- format. We investigated the expression pattern of commonly used basal cell / myoepithelial markers, such as p63 (a p53-homologous nuclear protein) and cytokeratin 14 (KRT-14). Both markers are detectable significantly in both systems. Additionally, the epithelial cell adhesion molecule (EpCAM) and cytokeratin 8 (KRT8) were tracked only in the 3D-system and KRT8 was only partially expressed in the2D-format. Consequently, 3D-platfrom in an organotypic setting expressed common breast tissue, luminal, and basal markers. Such mammary like organoids express human breast specific proteins including CSN2 (casein beta), milk protein peptides, and hormone receptors. The luminal cells specifically express EpCAM, MUC1 , CD49F, GATA3, CK8, and CK18 while basal cells will specifically express CK14, a-smooth muscle actin and P63. Eventually EpCAM and CD49F double positive cells can be detected at an earlier progenitor stage between D10 and D35. Interestingly, CSN2 expression is only captured at the last time point (D42) of the 3D-organotypic system and not in the 2D-directed differentiation platform.

[0479] Analysis of the mammary like organoids secretome showed secretion of human milk specific bioactives including oligosaccharides (including lactose and some HMOs), lipids (including 4 fatty acids), proteins (7 detected including caseins), and miRNA (75 detected, including 11 typically detected in HBM) as below described. Primary cell supernatant was analyzed for presence of lactose or human milk oligosaccharides following the procedure described in "Austin and Benet, Quantitative determination of non-lactose milk oligosaccharides, Analytica Chimica Acta 2018, 1010, 86-96” with minor modification. The samples were analysed with UHPLC and detected lactose or human milk oligosaccharides (HMOs) were quantified against a calibration curve of lactose and a mix of 7 HMOs (2'FL, 3FL, DFL, LNT, LNnT, 3'SL and 6’SL). The method had an estimated limit of 0.1 mg / L. In the primary cell supernatants, Lactose (0.22 mg / l) and 6'SL (0.32 mg / l) were detected at day 42.

[0480] Fatty acids were analysed in media and cell supernatants by gas chromatography coupled with flame ionization detector. Briefly, the supernatants obtained at day 42 is analysed to investigate the presence of fatty acids contained in several lipid classes. A 7890A gas-chromatograph with a 7693 autosampler with preparative station module equipped with a fused-silica CP-Sil 88 capillary column (100% cyanopropylpolysiloxane; 100 m, 0.25 mm id, 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. Preparation of FAMEs (fatty acids methyl esters) is performed by direct transesterification of sample with methanolic chloridric acid. Separation of FAMEs is performed using capillary gas chromatography-FID (GC). Identification of FAMEs is done by retention time (RT) and comparison with an external standard. Quantification of fatty acids is done by calculation using methyl C11 :0 as internal standard. Transesterification performance of the method is controlled with TAG C13:0 as second internal standard. After addition of internal standards, the solution was mixed with 2 mL of methanol, 2 mL of Methanol / HCI (3N) and 1 mL of hexane. After heating at 100°C / 60min, the sample is cooled down to room temperature (about 15 min) and the reaction is stopped by adding 2mL of water. After centrifugation the organic phase is directly injected into the GC.

[0481] Fatty acid results from protocol of Example 4a at time day 42 are reported in table 2 (differences observed between media and supernatant).

[0482] The table 2 below lists the expressed fatty acids in cell supernatant sample.

[0483] Proteins in the cell supernatant were analysed using SDS-PAGE profiling and then band isolation for identity confirmation by LC-MSMS. For SDS-PAGE analysis, the total volume of the prepared sample was loaded on the gel. A human milk sample was added for comparison as control. Selected gel regions (bands) were cut to look for human proteins by LC-MSMS. Eventually, bands were submitted to in-gel trypsin digestion and analyzed by LC-MSMS. LC-

[0484] MSMS data were analyzed with Peaks Studio and matched against the UniProt database for human proteins.

[0485] The table 3 below lists the best candidates for all the excised bands.

[0486] Exosome isolation and miRNA profiling was performed using ExoQuick polymer nets. ExoQuick polymer works to precipitate exosomes by forming a network and collects all exosomes of a certain size. Once the ExoQuick mesh is formed, a simple, low-speed centrifugation easily precipitates the exosomes as a pellet. The exosomes are intact, ready for protein or RNA analysis and are bioactive for functional studies. Precipitation buffer was added in a ration 0.25X to the sample then vortex. The mix was incubated overnight at 4°c. After incubation, samples were centrifuged 30 min at 1 ,500xg. The exosome pellet was re-suspended by vertexing in initial volume with Buffer XE (QIAGEN) for QC or Lysis Buffer from HTG EdgeSeq miRNA Whole Transcriptome Assay for miRNA profiling. In order to assess the extracellular vesicles (EVs) isolation, the supernatant was first centrifuged at 3000g for 15 min to remove cell pellet and debris. Then 100 microliters of media was used for an overnight precipitation at 4°c with ExoQuick buffer (ratio 0.25X). EV precipitates were recovered by centrifugation for 30 min at 1500g. Two precipitations were performed for each sample, one EV precipitation was resuspended in Buffer XE (QIAGEN) for potential further analysis, and a second one in only 50 ul HTG Lysis buffer in order to concentrate by 10-fold before miRNA profiling with HTG.

[0487] For miRNA profiling, samples were used directly in the first step of lysis. Thus, Whole sample was used directly and was lysed with Plasma lysis buffer in a ratiol :1 . Next, proteinase K (1 / 10) was added and the samples were incubated 3h at 50°c at 600rpm on Thermomixer. EVs were resuspended in Lysis buffer and lysed in the same conditions, with an incubation step at 95°c for 10min added before the lysis incubation. 26 l of lysate was process with 70 pl of oil on the HTG processor following the HTG EdgeSeq miRNA Whole Transcriptome Assay V2 procedure. For indexing and amplification libraries, samples were tagged with Illumina adaptors and indexes by PCR with OneTaq® Hot Start 2X Master Mix GC Buffer (95°C - 4 min; 16 cycles: 95°C-15 sec, 56°C-45 sec, 68°C- 45 sec; 68°C10 min; Hold at 4°C) and AMPure cleaned (ratio 2.5) on a robotic liquid handler SciClone NGS Workstation (Perkin Elmer). Pools were obtained with our custom pooling program on Hamilton robot. The samples were pooled based on GX touch Chip HS quantification. The pools were purified manually a second time with AMPure Bead (ratio 1.8) to remove potential remaining traces of primer-dimer and quantified with Qubit to adjust the final concentration to 2 nM. And as a last step, for MiSeq sequencing, pools were loaded on MiSeq at 20pM with a 5% PhiX spike and sequenced for 50 base Single read on MiSeq with 150V3 kit.

[0488] Briefly, 974 miRNAs detected in the in the cell supernatant which more than 75 of them are highly expressed miRNAs in the milk samples.

[0489] The table 4 below lists the top ten highly expressed miRNAs.

[0490] Our findings provide a novel iPSC-based 3D-organotypic model for studying the regulation and development of normal mammary cell fate and function as well as breast milk bioactives production.

[0491] Example 5 Cultivation and differentiation of hiPSCs into lactocytes to obtain a human milk like product using bone morphogenetic protein 4 (BMP4) and retinoic acid (RA)

[0492] In order to generate milk bioactives that closely resemble those found in human breast milk, different methods using bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) in 42 days and 31 days as a shortened time-course protocol were established in a 3D-platform using iPSCs derived mammary gland organoids as a biomimetic model of the human mammary gland (Figure 6).

[0493] 3D-iPSC cells in 42 days protocol and shortened time-course protocol (31 days -* describes shortened-modified protocols (31 days) including BMP4 and RA combinations) using 20 ng / mL of BMP4 and 1 piM of RA in single or combination format compared to the control condition can induce the expression of mammary gland progenitor markers such as EpCAM (CD326), CD49f, MUC1 (CD227) and GATA3 using flow cytometry quantification or maintain their expression profile during the differentiation time-course and / or pre-induction stages (Figure 7a-c) and post-induction period (Figure 7d-f).

[0494] We assessed the expression of different mammary epithelium markers in lactocytes derived from iPSCs in normal length and shortened protocols (Figure 8). This shortened protocol induces RELA as a specific gene for determining the lineage of the mammary epithelial cells during iPSC differentiation (Figure 8a). In addition to the mature luminal markers such as GATA3, specific lactocyte markers such as EpCAM, KRT8 / 18 are also induced in the shortened protocol (Figure 8b-e). In addition, when we switch to the 31 -day protocol, the ESRRA level is inducted (Figure 8f). As well, the shortened differentiation progress is associated with elevated levels of progenitor markers for mammary glands such as CD24 and ITGA6 (CD49f) (Figure 8g-h).

[0495] Human osteopontin peptides - GDSWYGLR and -YPDAVATWLNPDPSQK were specifically detected in the cell culture supernatant by LC-MS / MS (Figure 9).

[0496] Example 6

[0497] Analysis of human breast milk exosomes protein profile

[0498] A more systematic analysis of one specific human breast milk bioactive component, exosomes, as produced by Step A revealed very close profiles of composition with exosomes from human breast milk positive control.

[0499] Human iPSC were aggregated in 3D-culture using ultra-low binding 6-well plates with continuous rotation shacking to form embryoid bodies, and then subjected to a multi-steps differentiation protocol (41 days) to generate functional mammary gland organoids. The last 7 days of the differentiation protocol (from day 35 to day 41), the mammary gland organoids were subjected to a specific culture media supplemented with lactogenic hormones and growth factors to induce the secretion of milk-specific bioactive compounds. This media was renewed every other day (Day 35, Day 37, and Day 39).

[0500] Conditioned media was collected at Day 39 and Day 41 from step A, and pooled (which corresponds to the media that was in contact with the cells from Day 37 to Day 41). EVs were purified from the conditioned media and from skim human milk (commercially available at Lee Biosolutions, 991 -01 -P, collected from at least 5 lactating women after week 4 of lactation) by ultracentrifugation.

[0501] Analysis of the EV-associated protein profile by liquid chromatography - mass spectrometry (LC-MS) showed an overlap of approximately 30-50% when comparing breast milk control EVs and cell-based derived EVs (Figure 10a). Figure 10b shows that most of the proteins highly expressed in breast milk exosomes are also expressed in the cellbased exosomes including lactotransferrin (LTF), annexin A2 (ANXA2) and lactadherin (MFGE8), but also Mud, CD9, CD81, and CD63. Interestingly some proteins such as C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1, cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2), are not detected in cell-based

[0502] EVs but only in breast milk EV positive control.

[0503] Subsequent repetition of this experiment with 2 additional batches of cell-based EVs further confirmed that proteins Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1) are not detected in cell-based EVs but only in breast milk EV positive control. In addition, proteins C4a anaphylatoxin (CFA), Threonine— tRNA ligase 1, cytoplasmic (TARS1) and Cytosolic non-specific dipeptidase (CNDP2) were also not detected in one of the 2 additional cell-based EVs batches.

[0504] The proteomics analysis may be performed according to the following apparatus and conditions:

[0505] Analysis of the miRNA profile of EVs sequenced with HTG Molecular Whole Transcriptome Assay showed strong 95- 99% overlap between breast milk and cell-based exosomes (Figure 11a), including detection of already validated miRNA such as miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126 (Figure 11c). The results obtained from subsequent repetition of this experiment with 2 additional batches of cell-based EVs are shown in Figure 11b.

[0506] Analysis of the lipid fractions with the Lipometrix method (LC-MS / MS untargeted lipidomic analysis) in EVs purified from both conditioned media and human milk revealed similar overall profiles of lipid classes including cholesterol esters, ceramides, triacylglycerides, diacylglycerides, total phospholipids, alkanyl phospholipids, and alkenyl phospholipids (Figure 12). Same observation when analyzing subclasses profiles as phospholipid profile.

[0507] Flow cytometry analysis of the isolated EVs either from conditioned media (StepA) or from breast milk control showed a similar size ranging from 65 nm to 75nm (Figure 13).

[0508] Example 7

[0509] Human milk and lactocyte exosomes promote neuronal maturation in vitro

[0510] An in vitro model of neuronal maturation was used to assess the impact of supplementation with human milk EVs (extracellular vesicles / exosomes) or lactocyte (Lacto) EVS, for example obtained in accordance with Examples 5 and 6, on neuronal development using two distinct approaches.

[0511] In a first experiment, rat primary cortical neurons were exposed to human milk EVs or Lacto EVS, while in the second experiment, rat primary cortical neurons were treated with an astrocyte conditioned medium derived from rat primary cortical astrocytes pre-treated with human milk EVs or Lacto EVs.

[0512] In both cases, a multielectrode array (MEA) technology was used to record spontaneous electrical activity, allowing to assess neuronal activity and network maturation during the in vitro maturation process.

[0513] Preliminary findings show a significant enhancement in neuronal maturation in the presence of both human milk EVs or Lacto EVs, as compared to control conditions, as shown by an increased neuronal network activity.

[0514] Methods & Experimental design

[0515] 1. Cell culture

[0516] Rat Cortical Neurons (A1084002; Thermofisher) were seeded at a concentration of 20 000 cells / well in a volume of 300 pl onto the surface of 48-well MEA plates (M768-tMEA-48B_ref: 1000584, Axion Biosystem, 16 electrodes / well) that had been precoated with Poly D Lysine PDL (A38904-01; GIBCO). They were cultured in a neuronal medium: neurobasal (21103-049; GiBCO) supplemented with B27+ (2%, A35828-01; GIBCO), Glutamax (35050; GIBCO) and penicillin / streptomycin solution (1%, P0781; Sigma). The MEA plates were incubated at a temperature of 37°C and a 5% CO2 in a dedicated incubator.

[0517] Internalisation experiment: Rat Cortical Neurons were seeded at a concentration of 20 000 cells / well in a volume of 300 ul onto the surface of a 96-well plate that had been precoated with Poly D Lysine PDL. They were cultured in a neuronal medium consisting of neurobasal supplemented with B27+ (2%), Glutamax and penicillin / streptomycin solution (1%). At day in vitro (DIV) 2, 9 and 16 neurons were treated with CFSE conjugated human Milk EVs (100 exosomes / celt} and Lacto EVs (100 exosomes / celt) diluted in 30 ul of PBS. Treatment with vehicle (PBS) was used as control. 24h after treatment, the neurons were washed 5 x 30 sec with neuronal media and 1x30 sec with PBS. Cells were fixed with 4 % PFA (diluted in PBS) for 20 min on ice. Results are shown on Figure 15). All images were acquired using a confocal microscope (Leica confocal Microscope, SP8, Germany). Rat cortical Astrocytes (510086; Thermofisher) were seeded in 12 wells plates (353043; Falcon_Fisher), at a concentration of 3.104cells / well. They were cultured in DMEM complete (Dulbecco's Modified Eagle medium, 41966- 029; GIBCO) supplemented with FBS-Fetal Bovine Serum (15%, 10082147; GIBCO) and penicillin / streptomycin solution (1 %, P0781 ; Sigma).

[0518] Internalization experiment: Rat cortical Astrocytes were seeded in 12 wells plates (353043; Falcon_Fisher) covered with 15 mm cover slips that had been precoated with Poly D Lysine PDL. Astrocytes were seeded at a concentration of 1.104cells / well. They were cultured in DMEM complete supplemented with FBS-Fetal Bovine Serum (15%) and penicillin / streptomycin solution (1 %). At 80% of confluency, astrocytes were washed with (PBS) and astrocyte culture medium was replaced by fresh complete neuronal media {neurobasal supplemented with B27+, Glutamax and penicillin / streptomycin) containing CFSE conjugated human Milk EVs {100 exosomes / cell) and Lacto EVs {100 exosomes / cell) diluted in 30 ul of PBS. Treatment with vehicle (PBS) was used as control. 24h after treatment, the astrocytes were washed 5 x 30 sec with neuronal media and 1x30 sec with PBS. Cells were fixed with 4 % PFA (diluted in PBS) for 20 min on ice. Results are shown on Figure 14A and Figure 14B). All images were acquired using a confocal microscope (Leica confocal Microscope, SP8, Germany).

[0519] 2. Immunocytochemistry

[0520] Both neuronal and astrocyte cultures were washed 3 times in PBS 0.1 M. Nonspecific binding was blocked with 1% Triton-X10 (Sigma 93443) and 5% normal donkey [normal goat serum (Sigma G9023) or normal donkey serum (Sigma S30M), depending on the secondary antibody] in PBS (Gibco D8537). Wells were then incubated overnight at 4°C with the primary antibodies described below. Then incubated for 1 h with the corresponding secondary antibodies in PBS. 4,6 diamidino-2-phenylindole (DAPI) was used to reveal nuclei.

[0521] Primary antibodies used for immunocytochemistry were as follows: rabbit anti-GFAP (1 :200, DAKO _Z0334) for astrocytes and mouse anti-Beta III tubulin (1 :500. Abeam ab14545) for neurons.

[0522] Secondary antibodies were used as follows: goat anti-mouse Alexa-555 (1 : 1000, A21424_Thermo Fisher) and donkey anti-rabbit Alexa-555 (1 :1000, A-31572_Thermo Fisher).

[0523] Results are shown on Figures 14A and 14B and on Figure 15. All images were acquired using a confocal microscope (Leica confocal Microscope, SP8, Germany).

[0524] 3. Experimental conditions a. Treatment with EVs: At DIV 2, 9 and 16 neurons were treated with human Milk EVs (at three different concentrations: 100 exosomes / cell; 1.103exosomes / cell; 1.104exosomes / cell) and Lacto EVs {100 exosomes / cell) diluted in 30 ul of PBS. Treatment with vehicle (PBS) was used as control. This is shown on Figure 16. b. Treatment with astrocyte conditioned medium: At DIV 2 / 4 / 7 / 9 / 11 / 13 neurons were put in presence of astrocytic conditioned media (150 ml, 50% of the total volume per well) produced by astrocytes previously treated by Human Milk EVs or Lacto EVs. This is shown on Figure 17.

[0525] Preparation of astrocytes conditioned medium: Step 1 : At 80% of confluency, astrocytes were washed with (PBS) and astrocyte culture medium was replaced by fresh complete neuronal media (neurobasal supplemented with B27+, Glutamax and penicillin / streptomycin) further containing 250.106human-milk EVs (representing 1000 EVs per astrocyte), 2.5.109human-milk EVs (representing 10000 EVs per astrocyte) or 250.106Lacto EVs (representing 1000 EVs per astrocyte) in PBS. The vehicle (PBS) was used as control.

[0526] Step 2: Following a 48-hour treatment period, astrocytes were washed to eliminate any residual EVs. Subsequently, fresh neuronal medium was added onto the astrocytes to collect released factors and generate astrocyte conditioned media. At this stage, there are no EVs present in the medium.

[0527] Step 3: After 48h of treatment, astrocyte conditioned medium was then collected, filtered (0.2 pm, 10002261 ; Fisher) to remove cellular debris and immediately frozen at -80°C until being used for neuronal treatment. This is shown on Figure 18.

[0528] 4. MEA Recording and data processing

[0529] Extracellular recordings of neuronal spontaneous activity were obtained using the Axion Maestro Pro microelectrode array (MEA) systems. Data acquisition and analysis were performed with the Axion Integrated Studio (AxIS) Navigator 3.7.

[0530] From DIV 8 to DIV 19, the 48-well MEA plates (M768-tMEA-48B_ref: 1000584, Axion Biosystem, 16 electrodes / well) were placed into the Maestro pro and allowed at least 10 min to equilibrate, after which 10 min of spontaneous activity was recorded. All recordings were conducted at 37 °C and 5% CO2.

[0531] 5. MEA data analysis

[0532] Data analysis was performed in Axis Navigator 3.7, Neural module. A dataset of recordings collected between DIV 8 and 19, was pre-processed to remove inconsistent and low-quality recordings. Recordings with a minimum spike rate for Active Electrode > 5 spikes / min were included for analysis. Wells containing less than 2 active electrodes were also discarded.

[0533] Nine main groups of electrophysiological features were analyzed to characterize the spontaneous activity and maturation of cortical neurons in vitro: number of active electrodes, number of spikes, mean firing rate, number of bursts, bursts frequency, number of networks bursts, network burst frequency, synchrony index and the resistance. The table 5 below provides the electrophysiological features included in the analysis measured by MEA technology to assess neuronal maturation in vitro for neuronal activity and neuronal maturation:

[0534] MEA readouts and relevance for neuronal maturation

[0535] Number of Spikes: The total number of action potentials (spikes) detected over the duration of the analysis (10 min in this study). This readout indicates the overall level of spontaneous neuronal activity. As neurons mature, the number of spikes typically increases, reflecting the development of functional synapses.

[0536] Mean firing rate refers to the frequency at which neurons generate spikes. It is measured as the total number of spikes per unit of time (10 minutes in this study). This measurement provides additional information about the level of neuronal activity. The mean firing rate of neurons increases and stabilizes as they mature, reflecting the development of functional synaptic connections and neuronal network.

[0537] Synchrony index refers to the level of simultaneous spiking activity between electrodes, within the well. In vitro, this is referred to as the culture's synchrony. Synchrony index provides a quantitative measure of how well the neural activity is coordinated across different electrodes in the MEA, with values closer to 1 indicating higher synchrony.

[0538] This parameter informs on the following:

[0539] ■ Neural Communication: High synchrony during bursts suggests effective synaptic communication and network connectivity.

[0540] ■ Neural Health and Function: Patterns of synchrony and bursting can provide insights into the health and functionality of neural networks.

[0541] ■ Neuronal maturation: o Early Development: In the early stages of neuronal development, the synchrony index is usually low. Immature neurons are still forming synapses and establishing communication pathways. The spiking activity is more random and less coordinated. o Intermediate Stages: As neurons mature, they form more synaptic connections, and the network begins to exhibit more coordinated activity. This stage is characterized by the establishment of functional neural circuits and the beginning of synchronized spiking patterns. o Mature Neuronal Networks: In mature neuronal cultures, the synchrony index is higher, reflecting well- established and efficient communication pathways. Number of burst events detected over the duration of the analysis. A burst is a rapid series of spikes occurring within a short time.

[0542] Bursts represent periods of synchronized neuronal firing, which reflects information processing in the brain. The number of bursts can provide insights into the dynamics of neuronal networks and their ability to generate coordinated activity (neuronal network dynamics).

[0543] Burst Frequency: The total number of bursts divided by the duration of the analysis, expressed in Hertz (Hz).

[0544] This parameter informs on the following:

[0545] ■ Neuronal Excitability: Burst frequency provides a measure of how often neurons engage in synchronized firing events.

[0546] ■ Functional State: Changes in burst frequency can indica

[0547] ■ the alterations in the functional state of the neuronal network

[0548] The resistance (ohms, Q) quantifies the presence of cells on the electrode. To measure resistance, small electrical signals are delivered to the electrodes, and the ease with which the signal passes through the electrode-cell interface is measured. When the electrode is uncovered, electrical signal easily passes, and the resistance is low. When cells cover the electrode, less electrical signal passes and resistance is high. When cells die or detach, the resistance decreases.

[0549] 6. Statistical analysis

[0550] To facilitate the combination of multiple experiments, data from each time point was normalized as a percentage relative to its respective untreated control. Then the experiments were pooled together. The recordings were grouped in three intervals: DIV 8-11 (early stage), DIV 12-15 (intermediate stage), DIV 16-19 (late stages) (Figure 19).

[0551] Statistical analyses were performed using Prism 8.0 (GraphPad Software, Inc, https: / / www.qraphpad.com). A two-way repeated measures ANOVA was performed followed by Fisher's LSD post hoc multiple comparison test. Summary data and graphs were presented as mean ± SEM. Significance levels were defined at P < 0.05.

[0552] 7. Results

[0553] The bioavailability of EVs in both neurons and astrocytes in vitro was assessed. The results suggest that, in the presence of EVs, astrocytes and neurons engage in a complex process characterized by the internalization of EVs. This uptake, leading to the internalization of EVs and their content, might have a significant impact on cellular physiology via the activation of intracellular signaling pathways involved in cellular metabolism, membrane composition, neuronal excitability and / or astrocytic secretum composition.

[0554] The spontaneous activity of cortical neurons in culture was recorded with MEA from DIV 8 to DIV 19. Extracellular electrical signals were recorded and 9 features of spikes, bursts, synchrony, and resistance (see Tables 6 and 7) were analyzed post hoc to characterize the neuronal network activity.

[0555] Astrocyte conditioned media following treatment with EVs

[0556] Results obtained from the MEA technology (shown in Table 6 below and in Figures 20 and 21) suggest that conditioned medium from astrocytes, previously treated with EVs from Human Milk and Lacto EVs, enhances neuronal electrical maturation and network connectivity compared to untreated astrocyte conditioned medium. This is shown by a significant increase in total number of spike and mean firing rate, total number of bursts and burst frequency, as well as total number of network bursts at the later stage of development, during the 10 minutes of recording. However, there is no significant difference in resistance. Notably, Lacto EVs seem to be more effective than Human Milk EVs, showing greater efficacy at a lower concentration (1000 vs. 10000 EVs I astrocytes). Furthermore, the increase in synchrony observed with Lacto EVs, in the early stage of maturation, may indicate an accelerated neuronal network maturation.

[0557] In the context of brain maturation, these results suggest a positive impact on neuronal electrical activity and network connectivity. The significant increase in spike and mean firing rate indicates enhanced neuronal excitability. Moreover, the significant increase in burst and burst frequency points to increased synchronized firing of neuronal networks, which is important for information processing and learning. The significant increase in network burst further supports the idea of improved network connectivity and communication between neurons.

[0558] Overall, the trend of increased neuronal activity and network connectivity is promising and suggest a positive impact on in vivo brain maturation and increased brain connectivity. Table 6: f: Represent an increase when compared to the control.

[0559] Direct treatment of primary rat cortical neurons with EVs According to the findings from the MEA technology, there is a notable increase in total number of spikes and mean firing rate (Table 7 below and in Figures 22 and 23). The data also indicates that, compared to control conditions, treatment with both human milk and Lacto EVs enhances neuronal electrical excitability for intermediate and late stages of maturation.

[0560] Additionally, the increase in synchrony observed, at early stage of neuronal maturation, with both EVs treatments, suggests an accelerated maturation of the neuronal network, in vitro. In the context of brain maturation, these results suggest a positive impact on neuronal electrical activity and network connectivity. The significant increase in total number of spike and mean firing rate suggests an enhanced neuronal spontaneous activity. Finally, the overall trend of increased neuronal activity and network connectivity is promising and suggest a positive impact on in vivo brain maturation.

[0561] Table 7:

[0562] D-values by Fisher's LSD post hoc multiple comparison test. P-values < 0.05 are highlighted in bold, f: Represent an increase when compared to the control.

[0563] Example 8 Human milk and lactocyte exosomes bioefficacy in the brain in vivo The experimental protocol is illustrated in Figure 24.

[0564] Material and methods

[0565] 1. Animals Male and female C57BL / 6J were housed with their mother under standard conditions and allowed access to food and water ad libitum. They were mated to obtain pups for the NMS (neonatal maternal separation) protocol. After birth, murine pups were isolated from their mother from the post-natal days P2 to P14, three hours per day. These mice were named NMS mice. HM (human milk) exosomes (EVs) and lactocyte-derived (Lacto) Evs (3.00E+07 per gram per day) were daily administered orally by pipet feeding form from post-natal days P2 to P14. Pups were then left with their mothers up to weaning. 6 mice were used for each group. Control mice received the same dose of vehicle (PBS). Mice were sacrificed at PND21. All in vivo experiments were performed following the ethical guidelines set out by the International Association for the Study of Pain (IASP), complied with the European Union regulation and were approved by ethics committees.

[0566] 2. RNA extraction

[0567] Approximately 10 to 20mg of tissue were put in a Lysing Matrix D tube (MP Biomedicals, Santa Ana, California, USA) and placed on ice. Then 600piL of lysis buffer (Agencourt RNAdvance Tissue Kit, Beckman Coulter, Indianapolis, Indiana, USA) was added. The tubes were firmly closed and agitated for 2x1 minute at 6000rpm on Precellys (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was then extracted using the RNAdvance Tissue Kit (Beckman Coulter, Indianapolis, Indiana, USA), starting with 400piL of lyzate following the providers' recommendations. Total RNA was quantified using the Quant-IT RiboGreen™ RNA Assay Kit (Invitrogen, Carlsbad, California, USA) on a FilterMax F3 (Molecular Devices, Sunnyvale, California, USA). Total RNA quality was assessed using Fragment Analyzer-96 with DNF-471-0500 Standard Sensitivity RNA Analysis Kit (Agilent Technologies, Santa Clara, California, USA).

[0568] 3. mRNA library preparation and sequencing

[0569] 10Ong of total RNA was used to generate the mRNA libraries using the Illumina® Stranded mRNA Prep, Ligation Kit (Illumina Inc., San Diego, California, USA) following the provider recommendations and specifically 12 cycles of PCR amplification. Libraries were quantified with the Quant-IT Picogreen (Invitrogen, Carlsbad, California, USA) on a FilterMax F3 (Molecular Devices, Sunnyvale, California, USA). Size pattern was controlled with Fragment Analyzer-96 with DNF-474-0500 High Sensitivity NGS Fragment Analysis Kit (Agilent Technologies, Santa Clara, California, USA). Libraries were pooled at an equimolar ratio at a concentration of 48 nM. Sequencing was done with a loading concentration of 650 pM with a P4 300 kit for 2x150 cycles on an Illumina NextSeq 2000 (Illumina Inc., San Diego, California, USA), following the provider's recommendations.

[0570] 4. Analysis

[0571] Raw counts were obtained from sequences by mapping to the mouse reference genome (GRCm38-101) using STAR v.2.5.3 (Dobin A, Bioinformatics (Oxford, England), 29(1), 15-21 , 2012, which is hereby incorporated in its entirety) and counting using htseq-count v.0.6.1 (Anders S, Bioinformatics, 31 (2), 166-169, 2014, which is hereby incorporated in its entirety). A filter was applied to select only genes with a minimum of 5 reads in at least 5 samples (corresponding to a threshold of 0.165 on the CPM values) and non-annotated genes were discarded. 17838 features were kept with these filtering criteria. Quantro R package (Hicks SC et al., Genome Biology, 16(117), 2015, which is hereby incorporated in its entirety), which performs an anova-like test for global differences between groups of distributions, was used to assess whether all samples could be normalized together. Normalization was performed using qsmooth (smooth quantile normalization) (Hicks SC, et al., Biostatistics, 19(2), 2018, which is hereby incorporated in its entirety), a generalization of quantile normalization, which computes a weight at every quantile and compares the variability between groups relative to within groups. Differential expression analysis between groups was performed using edgeR (4.2.1) (Robinson, Bioinformatics (Oxford, England), 26(1), 139-140, 2010, which is hereby incorporated in its entirety) which estimates gene-wise negative binomial dispersions by calculating the adjusted profile log-likelihood for each gene and maximizing it by weighted likelihood empirical Bayes. A quasi-likelihood negative binomial generalized log- linear model was fitted to count data, and a gene-wise empirical Bayes quasi-likelihood F-test was conducted for each comparison. The resulting p-values were adjusted for multiple testing using the Benjamin! and Hochberg correction method (BH).

[0572] Results

[0573] 1 . Cortex Exposure of oral administration of HM EVs (Figure 25 and Table 8) and Lacto EVs (Figure 26 and Table 9) triggered a significant increase in the expression of genes included in myelination pathways hallmarks. Axon Ensheathment & Myelin Assembly: These processes mark the wrapping of axons by oligodendrocytes, forming the myelin sheath that insulates axons, increases conduction velocity, and supports the structural organization of cortical circuitry. This is essential for neuronal function, brain connectivity, efficient synaptic transmission and cognitive function (Xin, W et al., Nature, 633(8031), 856-863, 2024; Nicholson, M. et al., The European journal of neuroscience, 56(12), 6099-6114, 2022; Stassart, R. M. et al., Frontiers in neuroscience, 12, 467, 2018, each hereby incorporated in their entirety).

[0574] Oligodendrocyte Development and Differentiation: Elevated expression of genes and pathways underlying oligodendrocyte maturation indicates ongoing production of myelinating cells responsive to developmental cues and neural activity. This dynamic process allows adaptation and refinement of cortical circuit connectivity (Nicholson, M. et al., The European journal of neuroscience, 56(12), 6099-6114, 2022; Orthmann-Murphy, J. et al., eLife, 9, e56621 , 2020, each hereby incorporated in their entirety).

[0575] Taken together, these results suggest that exposure to the exosomes derived from both milk and lactocytes promotes, in an equivalent manner, key signaling pathways essential for brain function and myelination process. The upregulation of pathways related to oligodendrocyte differentiation, myelin assembly, and ensheathment of neurons indicates enhanced myelination processes. Myelination is crucial for the proper functioning of neurons, as it facilitates faster signal transmission along axons.

[0576] Increased GOBP activity for myelination reflects developmental transitions where cortical circuits shift from high plasticity to increased stability and functional specialization. It indicates region-specific maturation: deeper layers may mature earlier, contributing to output and computation differences across cortical hierarchies.

[0577] Abnormalities or deficits in these processes can impact cognitive function, as seen in conditions like multiple sclerosis, where disrupted myelination produces cortical disorganization and cognitive decline.

[0578] 2. Hippocampus

[0579] Exposure of oral administration of HM EVs (Figure 27 and Table 10) and Lacto EVs (Figure 28 and Table 11) triggered a significant increase in the expression of genes included in myelination.

[0580] Myelin Sheath in Hippocampal Neurodevelopment: Enhanced myelination in the hippocampus is linked to increased neuronal efficiency and better electrical conduction and supports faster action potential conduction, essential for developing efficient neural circuits. Activity-driven increases in myelin thickness and coverage indicate robust neurodevelopmental processes supporting learning and memory. Myelin sheath expansion— whether in thickness or in the proportion of ensheathed axons— suggests that axons are maturing properly and integrating well into hippocampal networks (Mitew, S. et al., Nature communications, 9(1), 306, 2018; Zhang, N. et al., Frontiers in neuroscience, 19, 1618468, 2025; Khelfaoui, H. et al., Cellular and molecular life sciences : CMLS, 81 (1), 181, 2024; Chen, J. F. et al., Neuron, 109(14), 2292-2307. e5, 2-21, each hereby incorporated in their entirety).

[0581] Postsynaptic Ensheathment Significance: Postsynaptic ensheathment, possibly involving glial processes enveloping dendritic spines or synapses, reflects the stabilization and maturation of synapses in development. Enhanced postsynaptic ensheathment may be associated with more efficient synaptic plasticity, maintenance of optimal circuitry function, and strengthened neuron-glia interactions— crucial for hippocampal development. Studies show that disruptions in these processes impair hippocampal circuit maturation and synaptic assembly, highlighting their importance in normal neurodevelopment (Ingvild Lynneberg Glaerum et al., Persistence of Cajal-Retzius cells in the postnatal hippocampus is required for development of dendritic spines of CA1 pyramidal cell bio xiv 2022.05.09.491146; doi: https: / / doi.Org / 10.1101 / 2022.05.09.491146; Liu, X. et al., Frontiers in molecular neuroscience, 14, 729273, 2021; Yang, Y. et al., Brain sciences, 14(4), 382, 2024, each hereby incorporated in their entirety).

[0582] Taken together, these results suggest that exposure to the exosomes derived from both milk and lactocytes promotes, in an equivalent manner, key signaling pathways essential for promoting a robust and coordinated maturation of hippocampal circuits, facilitating higher-order cognitive functions such as learning and memory.

[0583] They reflect adaptive neurodevelopment with greater capacity for activity-dependent plasticity, resilience against neurodevelopmental disorders, and potential for cognitive flexibility.

[0584] Exposure of oral administration of HM EVs (Figure 29 and Table 12) and Lacto EVs (Figure 30 and Table 13) triggered a significant increase in the expression of genes included in mitochondrial pathways hallmarks.

[0585] Mitochondrial Protein Complexes and Hippocampal Neurodevelopment. During neurodevelopment, neuronal cells in the hippocampus undergo a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS), highlighted by higher expression of mitochondrial respiratory complexes, such as the respirasome (supercomplexes of Complex I, III, and IV). Increased expression of these complexes, especially post-mitotic neuron differentiation, facilitates greater ATP production, which is vital for synapse formation and neuronal connectivity (Son, G. et al., BMB reports, 51 (11), 549-556, 2018; Jacobs, R. A. et al., Communications biology, 4(1), 938, 2021; Goyal M.S. et al., Cell Metabolism, 19(1), 49-57, 2014, each hereby incorporated in their entirety).

[0586] Impact on Aerobic Respiration and Bioenergetics: Enhanced mitochondrial content and higher activity of complexes like cytochrome c oxidase (Complex IV) are observed during critical time periods of hippocampal development, coinciding with increased energy demand for neuronal growth and synaptic activity. The strength and plasticity of synapses, major drivers of cognitive function, are tightly linked to mitochondrial ATP output— dependent on sufficient levels of these inner membrane protein complexes (Bulow, P. et al., iScience, 25(9), 104920, 2022; Jacobs, R. A. et al., Communications biology, 4(1), 938, 2021 , each hereby incorporated in their entirety).

[0587] Oxidative Phosphorylation, ATP Synthesis, and Electron Transport: Upregulation in protein synthesis and incorporation into the electron transport chain leads to improved mitochondrial respiration and synaptic function. If mitochondrial complex expression or function is impaired, neurodevelopmental processes suffer, contributing to synaptic dysfunction and linking to neurodevelopmental disorders (Bulow, P. et al., iScience, 25(9), 104920, 2022; He, K. et al., Frontiers in aging neuroscience, 14, 772066, 2022, each hereby incorporated in their entirety).

[0588] Gene Regulation and Synaptic Plasticity. Regulatory factors like NRF-1 and PGC-1o mediate the expression of mitochondrial respiratory complex genes; changes in their levels correlate with changes in hippocampal respiratory complex protein content, further impacting synaptic plasticity and memory formation. Activities requiring long-term potentiation or synaptic remodeling in the hippocampus are especially reliant on prompt upregulation of these gene products to meet dynamic energy needs (Biilow, P. et al., iScience, 25(9), 104920, 2022; Jacobs, R. A. et al., Communications biology, 4(1), 938, 2021 ; He, K. et al., Frontiers in aging neuroscience, 14, 772066, 2022, each hereby incorporated in their entirety).

[0589] Altogether, the increase in these mitochondrial complexes underpins both the energetic and signaling requirements for proper hippocampal neurodevelopment, supporting neuronal differentiation, synaptic maturation, and plasticity essential for learning and memory.

[0590] 1. Cerebellum

[0591] Exposure of oral administration of HM EVs (Figure 31 and Table 14) and Lacto EVs (Figure 32 and Table 15) triggered a significant increase in the expression of genes included in synapse formation and neuropeptide signaling pathways hallmarks.

[0592] Neuropeptide Signaling Pathway. The neuropeptide signaling pathway modulates neuronal activity, survival, differentiation, and synaptic communication throughout neurodevelopment. Elevation in this pathway often marks periods of active neural circuit formation, acting on target neurons through G protein-coupled receptors to influence neuronal differentiation, synapse assembly, and the formation of functional neural networks (Yeo, X. Y. et al., Biomedicines, 10(2), 343, 2022; Shi, Y. et al., EMBO reports, 23(8), e53267, 2022, each hereby incorporated in their entirety). Synapse Pruning: An increase in synapse pruning suggests the brain is optimizing its neural circuitry by selectively removing excess or weak synapses formed during early development. This process is essential for refining neural connections, enhancing cognitive efficiency, and ensuring proper maturation of remaining synapses. Aberrant pruning is linked to neurodevelopmental disorders (Faust, T. E. et al, Nature reviews. Neuroscience, 22(11 ), 657-673, 2021 ; Sakai J., Proceedings of the National Academy of Sciences of the United States of America, 117(28), 16096-16099, 2020; Neniskyte, U. , & Gross, C. T. , Nature reviews. Neuroscience, 18(11), 658-670, 2017, each hereby incorporated in their entirety).

[0593] Postsynaptic Signal Transduction: Enhanced postsynaptic signal transduction activity denotes active changes in the composition and function of postsynaptic densities, crucial for the maturation of synaptic strength, plasticity, and learning capabilities. During neurodevelopment, such changes reflect the dynamic remodeling of synaptic profiles and underpin the establishment of mature, responsive neuronal circuits (Kaizuka, T., & Takumi, T., The European journal of neuroscience, 59(11), 2894—2914, 2024; Song, W. et al., Neuron, 36(1), 105-119, 2002, each hereby incorporated in their entirety).

[0594] Taken together, these results suggest that exposure to both milk and lactocytes EVs promotes, in an equivalent manner, key signaling pathways essential for promoting a coordinated maturation of neuronal networks. It denotes active periods of synaptic formation, elimination, and refinement, leading to the establishment of efficient and adaptive brain circuitry typical of healthy neurodevelopment. Disruptions in these pathways are commonly implicated in neurodevelopmental and neuropsychiatric disorders.

[0595] 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 can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

Claims1. An isolated human breast milk exosome, wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome, for use in promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a subject with sub-optimal brain development.

2. A population of exosomes for use in promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a subject with sub-optimal brain development, wherein the population comprises isolated human breast milk exosomes wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome.

3. A composition comprising (i) isolated exosomes or (ii) a population of exosomes comprising isolated exosomes for use in promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a subject with sub-optimal brain development, wherein the isolated exosomes are isolated human breast milk exosome wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome.

4. An isolated human breast milk exosome, wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome, for use in the treatment of a disease or disorder selected from a demyelinating disease, a neurodevelopmental disorder, a neurocognitive disorder, or a neuropsychiatric disorder.

5. A population of exosomes for use in the treatment of a disease or disorder selected from a demyelinating disease, a neurodevelopmental disorder, a neurocognitive disorder, or a neuropsychiatric disorder, wherein the population comprises isolated human breast milk exosomes wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome.

6. A composition comprising (i) isolated exosomes or (ii) a population of exosomes comprising isolated exosomes for use in the treatment of a disease or disorder selected from a demyelinating disease, a neurodevelopmental disorder, a neurocognitive disorder, or a neuropsychiatric disorder, wherein the isolated exosomes are isolated human breast milk exosome wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (AP0A4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome.

7. The exosome for use of claim 4, the population of exosomes for use of claim 5, or the composition for use of claim 6, wherein the disease or disorder is a demyelinating disease, optionally selected from: multiple sclerosis, neuromyelitis optica spectrum disorder, transverse myelitis, acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

8. The exosome for use of claim 4, the population of exosomes for use of claim 5, or the composition for use of claim 6, wherein the disease or disorder is a neurodevelopmental disorder, optionally selected from: attention- deficit / hyperactivity disorder, autism spectrum disorders, communication disorders, intellectual disability, motor disorders, and specific learning disorder.

9. The exosome for use of claim 4, the population of exosomes for use of claim 5, or the composition for use of claim 6, wherein the disease or disorder is a neurocognitive disorder, optionally selected from: Alzheimer's disease, frontotemporal lobar degeneration, Lewy body disease, vascular disease, traumatic brain injury, substance or medication use, HIV infection, prion disease, Parkinson's disease, and Huntington's disease.

10. The exosome for use of claim 4, the population of exosomes for use of claim 5, or the composition for use of claim 6, wherein the disease or disorder is a neuropsychiatric disorder, optionally selected from: seizure, attention deficit disorder, cognitive deficit disorder, palsies, uncontrolled anger, migraine headaches, addictions, eating disorders, depression, and anxiety.

11. Use of an isolated human breast milk exosome, wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome, for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a healthy subject.

12. Use of a population of exosomes for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a healthy subject, wherein the population comprises isolated human breast milk exosomes wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome.

13. Use of a composition comprising (I) isolated exosomes or (ii) a population of exosomes comprising isolated exosomes for promoting or supporting brain structure, brain connectivity, cognitive potential, learning potential, intellectual potential and / or cognitive functioning in a healthy subject, wherein the isolated exosomes are isolated human breast milk exosome wherein Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4) and Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (PLOD1) are not detected by liquid chromatography - mass spectrometry in the exosome.

14. The exosome for use of any one of claims 1 , 4, or 7 to 10, the population of exosomes for use of any one of claims 2, 5, or 7 to 10, the composition for use of any one of claims 3, or 6 to 10, the use of an exosome of claim 11 , the use of a population of exosomes of claim 12, or the use of a composition of claim 13, wherein C4a anaphylatoxin (CFA), Hypoxia up-regulated protein 1 (HYOU1), Apolipoprotein A-IV (APOA4), Procollagen-lysine,2-oxoglutarate 5- dioxygenase 1 (PLOD1), Threonine— tRNA ligase 1 , cytoplasmic (TARS1), and Cytosolic non-specific dipeptidase (CNDP2) are not detected by liquid chromatography - mass spectrometry in the exosome.

15. The exosome for use of any one of claims 1, 4, 7 to 10, or 14, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14, the composition for use of any one of claims 3, 6 to 10, or 14, the use of an exosome of claim 11 or claim 14, the use of a population of exosomes of claim 12 or claim 14, or the use of a composition of claim 13 or claim 14, wherein the exosome comprises lactotransferrin (LTF), annexin A2 (ANXA2) and lactadherin (MFGE8), MUC1 and tetraspanins CD9, CD81 and CD63.

16. The exosome for use of any one of claims 1 , 4, 7 to 10, 14, or 15, the population of exosomes for use of any one of claims 2, 5, 7 to 10, 14, or 15, the composition for use of any one of claims 3, 6 to 10, 14, or 15, the use of an exosome of any one of claims 11 , 14, or 15, the use of a population of exosomes of any one of claim 12, 14, or 15, or the use of a composition of any one of claims 13 to 15, wherein the exosome comprises one or more miRNAs selected from miR-148a-3p, miR-22-3p, miR-125b-5b and miR-6126.

17. The exosome for use of any one of claims 1 , 4, 7 to 10, or 14 to 16, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14 to 16, the composition for use of any one of claims 3, 6 to 10, or 14 to 16, the use of an exosome of any one of claims 11, or 14 to 16, the use of a population of exosomes of any one of claim 12, or 14 to 16, or the use of a composition of any one of claims 13 to 16, wherein the exosome comprises one or more lipids selected from cholesterol esters, ceramides, triacylglycerides, diacylglycerides, lyso-phospholipids, total phospholipids, alkanyl phospholipids, and alkenyl phospholipids.

18. The exosome for use of any one of claims 1 , 4, 7 to 10, or 14 to 17, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14 to 17, the composition for use of any one of claims 3, 6 to 10, or 14 to 17, the use of an exosome of any one of claims 11, or 14 to 17, the use of a population of exosomes of any one of claim 12, or 14 to 17, or the use of a composition of any one of claims 13 to 17, wherein the exosome comprises one or more phospholipids selected from sphingomyelins, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.

19. The exosome for use of any one of claims 1 , 4, 7 to 10, or 14 to 18, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14 to 18, the composition for use of any one of claims 3, 6 to 10, or 14 to 18, the use of an exosome of any one of claims 11, or 14 to 18, the use of a population of exosomes of any one of claim 12, or 14 to 18, or the use of a composition of any one of claims 13 to 18, wherein the exosome has a diameter of 50 to 100nm.

20. The exosome for use of claim 19, the population of exosomes for use of claim 19, the composition for use of claim 19, the use of an exosome of claim 19, the use of a population of exosomes of claim 19, or the use of a composition of claim 19, wherein the exosome has a diameter size of 65 to 75nm.

21. The exosome for use of any one of claims 1 , 4, 7 to 10, or 14 to 20, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14 to 20, the composition for use of any one of claims 3, 6 to 10, or 14 to 20, the use of an exosome of any one of claims 11, or 14 to 20, the use of a population of exosomes of any one of claim 12, or 14 to 20, or the use of a composition of any one of claims 13 to 20, wherein the exosome has the same function as exosomes derived from natural human breast milk.

22. The exosome for use of any one of claims 1 , 4, 7 to 10, or 14 to 21, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14 to 21 , the composition for use of any one of claims 3, 6 to 10, or 14 to 21 , the use of an exosome of any one of claims 11, or 14 to 21, the use of a population of exosomes of any one of claim 12, or 14 to 21 , or the use of a composition of any one of claims 13 to 21, wherein the exosome has the same structure as exosomes derived from natural human breast milk.

23. The exosome for use according to any one of claims 1 , 4, 7 to 10, or 14 to 22, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14 to 22, the composition for use of any one of claims 3, 6 to 10, or 14 to 22, the use of an exosome of any one of claims 11, or 14 to 22, the use of a population of exosomes of any one of claims 12, or 14 to 22, or the use of a composition of any one of claims 13 to 22, wherein the exosome or population of exosomes is obtainable according to an in vitro method comprising:A) Generating lactocyte mammary-like gland organoids derived from human induced pluripotent stem cells (hiPSC),B) Secreting a human milk like product from said lactocytes, andC) Purifying the exosomes from the human milk like product to remove impurities, optionally by chromatography or filtration or ultracentrifugation, in order to isolate the exosomes, wherein step A) comprises culturing the hiPSCs in a culture medium comprising BMP4 and / or RA.

24. The exosome, the population of exosomes or the composition for use of claim 23, or the use of an exosome, of a population of exosomes or of a composition according to claim 23, wherein step A) is between a 30-day and 45- day process.

25. The exosome, the population of exosomes or the composition for use of claim 23 or 24, or the use of an exosome, of a population of exosomes or of a composition according to claim 23 or claim 24, wherein Step A) is conducted in 3D suspension culture conditions.

26. The exosome, the population of exosomes or the composition for use of any one of claims 23 to 25, or the use of an exosome, of a population of exosomes or of a composition according to any one of claims 23 to 25, whereinstep C) further comprises formulating the isolated exosomes into a powder form, optionally by spray drying or freeze drying, or liquid form.

27. The exosome, the population of exosomes or the composition for use of any one of claims 23 to 26, or the use of an exosome, of a population of exosomes or of a composition according to any one of claims 23 to 26, wherein step C) further comprises sterilising the isolated exosomes.

28. The exosome, the population of exosomes or the composition for use of any one of claims 23 to 27, or the use of an exosome, of a population of exosomes or of a composition according to any one of claims 23 to 27, wherein step C) further comprises storing the isolated exosomes at below freezing, optionally at -80°C.

29. The exosome, the population of exosomes or the composition for use of any one of claims 23 to 28, or the use of an exosome, of a population of exosomes or of a composition according to any one of claims 23 to 28, further comprising formulating the isolated exosomes with supplementary nutritional ingredients.

30. The exosome, the population of exosomes or the composition for use of any one of claims 23 to 29, or the use of an exosome, of a population of exosomes or of a composition according to any one of claims 23 to 29, wherein the isolated exosomes are dispensed into a container for consumption.

31. The exosome for use of any one of claims 1, 4, 7 to 10, or 14 to 22, the population of exosomes for use of any one of claims 2, 5, 7 to 10, or 14 to 22, the composition for use of any one of claims 3, 6 to 10, or 14 to 22, the use of an exosome of any one of claims 11, or 14 to 22, the use of a population of exosomes of any one of claims 12, or 14 to 22, or the use of a composition of any one of claims 13 to 22, wherein the isolated exosomes are in a powder form, optionally spray dried or freeze dried, or liquid form.

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