Multi-tissue organoid platform for developmental and disease modelling applications

Abstract

The invention relates to a chemically defined universal medium (CDUM) comprising one or more of: a) a first basal medium, such as Iscove's Modified Dulbecco's Medium (IMDM); b) a second basal medium comprising a nutrient mixture comprising amino acids, vitamins and inorganic salts; c) a polymer, such as poly(vinyl alcohol) (PVA); d) lipids; e) an activator of the ERK / MAPK pathway, such as insulin; f) an iron carrier; g) selenium supplement; h) a cell growth and proliferation promoter, such as ethanolamine; i) a first anti-oxidant, such as monothioglycerol; j) a second anti-oxidant, such as ascorbic acid-2-phosphate (AA2P); k) L-glutamine supplement; and related media and methods for iPSC differentiation into organoids.

Description

[0001] Multi-tissue organoid platform for developmental and disease modelling applications

[0002] The present invention relates to a medium composition and method for generating multi tissue organoids from hiPSCs. These organoids can be used in a variety of applications including developmental and disease modelling applications as well as drug discovery, toxicology and drug target validation and efficacy testing. These models maybe applied to basic research, translational pre-clinical applications or cell and tissue replacement therapies.

[0003] The pathophysiology of human disease is complex, involving genetics, environmental and lifestyle factors. At the tissue level, disease is driven by the complexity of alterations in cellular metabolism and cell-cell communication, genomic instability, changes in tissue cellular composition, remodelling of tissue architecture and extracellular matrix and changes in vascularisation, inflammation, and immune modulation.

[0004] Disease models that recapitulate these features have been the domain of animal models. While these models have delivered crucial insight into human development and disease, physiological, genetic, immune, and metabolic differences create significant challenges translating animal data to human.

[0005] Human tissue explant models along with primary and immortalised human cell lines offer human specificity however extensive use is limited due to access and supply issues, complex culture requirements, limited experimental life-span and donor-to-donor variability.

[0006] More recently, development of pluripotent stem cell (PSC)-derived multi-cell-type organoids and microtissues have been described that offer opportunities for more complex disease and developmental modelling. Typically these models are created using four different approaches whereby

[0007] PSCs are: 1) differentiated to the desired cell type and then mixed with primary derived cell types to create an organoid[29, 30]; 2) differentiated towards a primary germ layer that further spontaneously differentiates and self-organises into a 3D organoid; 3) co-differentiated into multiple cell types using protocols designed to introduce heterogeneity into the resulting population of cells which then further spontaneously differentiate and self-organise into complex organoids; 4) differentiated into specific cell types using separate differentiation platforms and then recombined in 3D allowing selforganisation into organoids. An additional methodology, that may combine any of the above approaches involves the assembly of multiple organoids structures into a mixed organoid conformation termed an "assembloid" that self-organises into a complex, multi-region organoid. While each approach produces organoids with variable cellular composition, complexity and functional profiles, each approach creates limitations in the versatility and application of the organoids. Organoids comprised PSC-derived and primary cells will exhibit batch-to-batch variability and may limit inclusion of some immune cell types, single progenitor-cell-derived tissues will be deficient in cell types derived from other germ layers rendering full tissue composition incomplete, organoids derived from the spontaneous differentiation of multiple germ layers will exhibit variability in the efficiency of germ layer and progenitor cell differentiation at the level of the cell line and protocol used and subsequently the downstream composition of organoids will amplify this variability.

[0008] Variability in spontaneously derived tissues has been demonstrated during the production of stem- cell-based embryo models where the expected structure is well defined and therefore easily scored against those produced in-vitro, in these models reproducibility is <1%

[0047] , While recombining individual populations of cells, derived from PCS allows for control of individual cell numbers and the cellular composition of microtissues, a limited number of cell types have been recombined as diverse medium and cell signalling requirements for each cell type complicate their eventual co-culture resulting in limited survival and functionality of individual cell types.

[0009] What is needed is a new platform that can generate multiple cell types, such as fibroblasts, endothelial cells, macrophages and dendritic cells from hiPSCs, using a single medium formulation.

[0010] Summary of Invention

[0011] According to a first aspect of the present invention, there is provided: a chemically defined universal medium (CDUM) comprising one or more of: a) a first basal medium, such as Iscove's Modified Dulbecco's Medium (IMDM); b) a second basal medium comprising a nutrient mixture comprising amino acids, vitamins and inorganic salts; c) a polymer, such as poly(vinyl alcohol) (PVA); d) lipids; e) an activator of the ERK / MAPK pathway, such as insulin; f) an iron carrier; g) selenium supplement; h) a cell growth and proliferation promoter, such as ethanolamine; i) a first anti-oxidant, such as monothioglycerol; j) a second anti-oxidant, such as ascorbic acid-2-phosphate (AA2P); k) L-glutamine supplement; and

[0012] I) optionally non-essential amino acids supplement.

[0013] The chemically defined universal medium may further comprise at least one antibiotic.

[0014] According to another aspect, there is provided the use of the chemically defined universal medium according to the invention for the differentiation of human induced pluripotent stem cells (hiPSCs) into a different cell type.

[0015] According to another aspect, there is provided the use of the chemically defined universal medium according to the invention for the differentiation of induced pluripotent stem cells (iPSCs) into a different cell type. As an alternative to human, the cell types may be non-human animal cells, such as non-human mammalian cells. The cell types may be mouse, rat, pig, of simian.

[0016] The invention advantageously provides a chemically defined, xeno-free, serum-free platform to generate various cell types, such as fibroblasts, endothelial cells, macrophages and dendritic cells from hiPSCs using a single medium formulation. The resulting cells display functional profiles consistent with each cell type and all cells remain viable and functional in the same medium formulation. These cells can be combined with hiPSC derived parenchymal cells to form organoids and microtissues representative of different organs and disease modalities. The platform advantageously allows for simple, reproducible and precise generation of microtissues that are tuneable along cellular, genetic, and environmental variables and contain cells important for modelling multiple modalities of disease progression and importantly supports inclusion of multiple autologous immune cell types. Cell types can include organoids with any parenchymal cell such as epithelium from the gut, lung, liver, cardiomyocytes with fibroblasts, endothelial cells, macrophages and dendritic cells. T-cells, NK cells, neuronal and muscle derivatives can also be added. These different populations of cells remain in the organoids at predefined ratios, but can also respond to stimuli to cause a range of cellular and tissue specific physiological effects. This platform addresses many of the limitations of current approaches to co-culture organoids and microtissues and will facilitate a greater understanding of the role epithelial, immune, stromal, and vascular cells play in progression of disease.

[0017] The First Basal Medium The first basal medium may comprise or consist of minimal essential medium (MEM). In one embodiment, the first basal medium comprises or consists of Iscove's Modified Dulbecco's Medium

[0018] (IMDM). The Iscove's Modified Dulbecco's Medium (IMDM) may be formulated as originally described in the journal article N.N. Iscove and F. Melchers (1978), J. Exper. Med., 147:923, which is incorporated herein in its entirety by reference. IMDM is routinely used in the art, either neat or diluted with other medium and / or supplements, as a medium for culturing cells. As such, the person skilled in the art will be familiar with IMDM. Alternatively, the first basal medium may comprise Dulbecco's Modified Eagle

[0019] Medium (DMEM), Dulbecco's Basal Medium Eagle (BME), or RPMI-1640.

[0020] The first basal medium, such as IMDM, may not comprise serum, such as foetal bovine serum. The first basal medium, such as IMDM, may not comprise lipids. The first basal medium, such as IMDM, may not comprise proteins. The first basal medium, such as IMDM, may not comprise iron. The first basal medium, such as IMDM, may not comprise growth factors. In one embodiment, the first basal medium. such as IMDM, does not substantially comprise serum (such as foetal bovine serum), lipids, proteins, iron, and growth factors. The first basal medium, such as IMDM, may or may not comprise Phenol

[0021] Red. The first basal medium, such as IMDM, may be xeno-free.

[0022] The first basal medium, such as IMDM, in the composition may be diluted by the other components of the chemically defined universal medium so that it comprises about 70% (v / v) of the chemically defined universal medium. In some embodiments, the first basal medium, such as IMDM, in the composition is diluted by the other components of the chemically defined universal medium so that it comprises about 70.32% (v / v) of the chemically defined universal medium. Alternatively, in these embodiments, the first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises from about 50% (v / v) to about 80% (v / v) of the chemically defined universal medium. Alternatively, in these embodiments, the first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises from about 60% (v / v) to about 80% (v / v) of the chemically defined universal medium. Alternatively, in these embodiments, the first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises from about

[0023] 65% (v / v) to about 75% (v / v) of the chemically defined universal medium. The first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises at least about 50% (v / v), at least about 55% (v / v), at least about 60% (v / v), at least about 65% (v / v), at least about 68% (v / v), at least about 69% (v / v), at least about 70% (v / v), at least about 71% (v / v), at least about 72% (v / v), at least about 75% (v / v), or at least about 80% (v / v), of the chemically defined universal medium. The first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises up to about 50%

[0024] (v / v), up to about 55% (v / v), up to about 60% (v / v), up to about 65% (v / v), up to about 68% (v / v), up to about 69% (v / v), up to about 70% (v / v), up to about 71% (v / v), up to about 72% (v / v), up to about

[0025] 75% (v / v), or up to about 80% (v / v), of the chemically defined universal medium.

[0026] In other embodiments, the first basal medium, such as IMDM, in the chemically defined universal medium is diluted by the other components of the chemically defined universal medium so that it comprises about 47% (v / v) of the chemically defined universal medium. In other embodiments, the first basal medium, such as IMDM, in the chemically defined universal medium is diluted by the other components of the chemically defined universal medium so that it comprises about 46.9% (v / v) of the chemically defined universal medium. Alternatively, in these embodiments, the first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises from about 35% (v / v) to about 60% (v / v) of the chemically defined universal medium. Alternatively, in these embodiments, the first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises from about

[0027] 40% (v / v) to about 50% (v / v) of the chemically defined universal medium. The first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises at least about 35% (v / v), at least about 40% (v / v), at least about 44% (v / v), at least about 45% (v / v), at least about 46% (v / v), at least about 47% (v / v), at least about 48% (v / v), at least about 49% (v / v), at least about 50% (v / v), at least about 55% (v / v), or at least about 60% (v / v), of the chemically defined universal medium. The first basal medium, such as IMDM, may be diluted by the other components of the chemically defined universal medium so that it comprises up to about 35%

[0028] (v / v), up to about 40% (v / v), up to about 44% (v / v), up to about 45% (v / v), up to about 46% (v / v), up to about 47% (v / v), up to about 48% (v / v), up to about 49% (v / v), up to about 50% (v / v), up to about

[0029] 55% (v / v), or up to about 60% (v / v), of the chemically defined universal medium.

[0030] The Second Basal Medium Comprising a Nutrient Mixture

[0031] The second basal medium may be a medium with a mixture of nutrients suitable to grow or maintain cells, such as iPSCs. The second basal medium may be a Ham's medium such as a Ham's nutrient mixture, preferably Ham's F-12. The Ham's F-12 (F12) may be formulated according to its standard composition as known in the art (e.g. https: / / www.therrnofisher,corn / uk / en / home / technical- resources / media-formuiation.64.htmi and 1. Ham, R. G. (1965) Proc. Nat. Acad. Sci., 53, 288, which is herein incorporated by reference). Nutrient mixture, such as F12, is routinely used in the art, either neat or diluted with other media and / or supplements, as a medium for culturing cells. As such, the person skilled in the art will be familiar with basal medium comprising a nutrient mixture, such as F12.

[0032] A skilled person will recognise that an equivalent medium to Ham's F12 may be provided, for example comprising the same or substantially similar components and / or quantities of component.

[0033] The nutrient mixture may comprise one or more, or all of, the amino acids selected from L-Asparagine,

[0034] L-Aspartic acid, L-Cysteine, L-Glutamic Acid, L-Glutamine, L-Histidine hydrochloride, L-lsoleucine, L-

[0035] Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-

[0036] Tyrosine, and L-Valine. Additionally, or alternatively, the nutrient mixture may comprise one or more, or all of, the vitamins selected from Biotin, Choline chloride, D-Calcium pantothenate, Folic Acid,

[0037] Niacinamide, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamin B12, and i-

[0038] Inositol. Additionally, or alternatively, the nutrient mixture may comprise one or more, or all of, the inorganic salts selected from Calcium Chloride, Cupric sulfate, Ferric sulfate, Magnesium Chloride,

[0039] Potassium Chloride, Sodium Bicarbonate, Sodium Chloride, Sodium Phosphate dibasic, and Zinc sulfate. Additionally, or alternatively, the nutrient mixture may comprise one or more, or all of, the components selected from D-Glucose (Dextrose), Hypoxanthine Na, Linoleic Acid, Lipoic Acid, Phenol

[0040] Red, Putrescine 2HCI, Sodium Pyruvate, and Thymidine.

[0041] In one embodiment, the nutrient mixture may comprise one or more, or all of, the components selected from L-Asparagine, L-Aspartic acid, L-Cysteine, L-Glutamic Acid, L-Glutamine, L-Histidine hydrochloride, L-lsoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-

[0042] Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Biotin, Choline chloride, D-Calcium pantothenate, Folic

[0043] Acid, Niacinamide, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamin B12, i-

[0044] Inositol, Calcium Chloride, Cupric sulfate, Ferric sulfate, Magnesium Chloride, Potassium Chloride,

[0045] Sodium Bicarbonate, Sodium Chloride, Sodium Phosphate dibasic, Zinc sulfate, D-Glucose (Dextrose),

[0046] Hypoxanthine Na, Linoleic Acid, Lipoic Acid, Phenol Red, Putrescine 2HCI, Sodium Pyruvate, and

[0047] Thymidine. The skilled person may omit or substitute 1, 2, 3, 4 or 5 of such components for the same nutrient function.

[0048] The second basal medium may be xeno-free. The second basal medium may not comprise foetal bovine serum. The second basal medium may comprise L-alanyl-L-glutamine, also known in the art as

[0049] GlutaMAX™. Alternatively, the second basal medium may comprise no L-alanyl-L-glutamine. In some embodiments, the second basal medium in the chemically defined universal medium is diluted by the other components of the chemically defined universal medium so that it comprises about 23%

[0050] (v / v) of the composition. In some embodiments, the second basal medium in the chemically defined universal medium is diluted by the other components of the chemically defined universal medium so that it comprises about 23.44% (v / v) of the chemically defined universal medium. Alternatively, in these embodiments, the second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises from about 15% (v / v) to about 30% (v / v) of the chemically defined universal medium. Alternatively, in these embodiments, the second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises from about 20% (v / v) to about 30% (v / v) of the chemically defined universal medium.

[0051] Alternatively, in these embodiments, the second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises from about 20% (v / v) to about 25% (v / v) of the chemically defined universal medium. The second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises at least about 15% (v / v), at least about 20% (v / v), at least about 21% (v / v), at least about 22% (v / v), at least about 23% (v / v), at least about 24% (v / v), at least about 25% (v / v), at least about 26% (v / v), or at least about 30% (v / v), of the chemically defined universal medium. The second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises up to about 15% (v / v), up to about 20% (v / v), up to about 21% (v / v), up to about 22% (v / v), up to about

[0052] 23% (v / v), up to about 24% (v / v), up to about 25% (v / v), up to about 26% (v / v), or up to about 30%

[0053] (v / v), of the chemically defined universal medium.

[0054] In other embodiments, the second basal medium in the chemically defined universal medium is diluted by the other components of the composition so that it comprises about 47% of the chemically defined universal medium. In other embodiments, the second basal medium in the chemically defined universal medium is diluted by the other cell medium components of the chemically defined universal medium so that it comprises about 46.9% of the chemically defined universal medium. Alternatively, in these embodiments, the second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises from about 35% to about 60% of the chemically defined universal medium. Alternatively, in these embodiments, the second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises from about 40% to about 50% of the chemically defined universal medium. Alternatively, in these embodiments, the second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises from about 45% to about 50% of the chemically defined universal medium. The second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises at least about 35% (v / v), at least about 40% (v / v), at least about 44% (v / v), at least about 45% (v / v), at least about 46% (v / v), at least about 47% (v / v), at least about 48% (v / v), at least about 49% (v / v), at least about 50% (v / v), at least about 55% (v / v), or at least about 60% (v / v), of the chemically defined universal medium. The second basal medium may be diluted by the other components of the chemically defined universal medium so that it comprises up to about 35% (v / v), up to about 40% (v / v), up to about 44% (v / v), up to about 45% (v / v), up to about 46% (v / v), up to about 47% (v / v), up to about 48% (v / v), up to about

[0055] 49% (v / v), up to about 50% (v / v), up to about 55% (v / v), or up to about 60% (v / v), of the chemically defined universal medium.

[0056] The Polymer

[0057] The polymer may be any polymer, natural or synthetic, that can function as a serum substitute. The polymer may be a synthetic polymer. The synthetic polymer may otherwise be termed as a non-natural polymer (i.e. cannot be found in nature), or a polymer derived from petroleum oil. The synthetic polymer may comprise polymers of average molecular weight of about 30,000-70,000 Da. In one embodiment, the synthetic polymer comprises or consist of poly(vinyl alcohol) (PVA, also known as poly(ethenol)). The polymer may comprise polymer molecules having an average molecular weight of about 30,000-70,000 Da. The poly(vinyl alcohol) (PVA, also known as poly(ethenol)) may comprise poly(vinyl alcohol) polymers of average molecular weight of about 30,000-70,000 Da.

[0058] The polymer, such as PVA, may initially be provided in the composition as a powder (e.g. as a hydrolysed compound) to be dissolved therein. Alternatively, the polymer, such as PVA, may be provided in the composition as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated polymer, such as PVA, solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline (PBS) or Dulbecco's PBS (DPBS).

[0059] Preferably, the polymer, such as PVA, is provided in the composition at a concentration of about 10 mg / mL. The polymer, such as PVA, may be provided in the composition at a concentration of from about 5 mg / mL to about 15 mg / mL. The polymer, such as PVA, may be provided in the composition at a concentration of at least about 5 mg / mL, at least about 6 mg / mL, at least about 7 mg / mL, at least about 8 mg / mL, at least about 9 mg / mL, at least about 9.5 mg / mL, at least about 9.75 mg / mL, at least about 10 mg / mL, at least about 10.25 mg / mL, at least about 10.5 mg / mL, at least about 11 mg / mL, at least about 12 mg / mL, at least about 13 mg / mL, at least about 14 mg / mL, or at least about 15 mg / mL.

[0060] The Lipids

[0061] The lipids may be chemically defined lipids, for example which may be added as a chemically defined lipid concentrate, such as described in Table 5 herein.

[0062] The lipids may comprise one or more, or all of, arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, pluronic F-

[0063] 68 (also known as, poloxamer 188, (C3H6O.C2H4O)x or polyoxyethylene-polyoxypropylene block copolymer), stearic acid, polysorbate 80 (e.g. Tween 80®). Such lipids may be provided as a concentrate in the proportions set out in Table 5 herein, or at least within 10% variance thereof.

[0064] The selected lipids may be provided in the composition as a solution, e.g. a concentrated pre-mix solution that is diluted in the composition. The lipid concentrate may be a lOOx concentrate, wherein the CDUM medium comprises about 0.1% v / v of the lOOx lipid concentrate. In such embodiments, the concentrated PVA solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline

[0065] (PBS) or Dulbecco's PBS (DPBS). The lipids may be at a concentration of at least about 2 mg / L in the medium. In another embodiment, the lipids may be at a concentration of at least about 5 mg / L in the medium. The lipids may be at a concentration of from about 2 mg / L to 50 mg / L in the medium. In another embodiment, the lipids may be at a concentration of from about 5 mg / L to 30 mg / L in the medium. In another embodiment, the lipids may be at a concentration of from about 5 mg / L to 20 mg / L in the medium. In another embodiment, the lipids may be at a concentration of from about 6 mg / L to 10 mg / L in the medium. In one embodiment, the lipids may be at a concentration of about 8 mg / L in the medium. The lipids may comprise one or more, or all of, arachidonic acid, cholesterol, DL- alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, pluronic F-68 (also known as, poloxamer 188, (C3H6O.C2H4O)x or polyoxyethylene- polyoxypropylene block copolymer), stearic acid, or polysorbate 80 (e.g. Tween 80®), wherein the lipids are at a total concentration of from about 6 mg / L to 10 mg / L in the medium.

[0066] The selected lipids may be sourced commercially. The selected lipids may comprise or consist of lipids that are normally found in animal serum, or synthetic equivalents or derivatives thereof. The Activator of the ERK / MAPK pathway

[0067] The activator of the ERK / MAPK pathway may comprise insulin, or an insulin equivalent that is capable of activating the same ERK / MAPK pathway as insulin, such as a small molecule. The activator of the

[0068] ERK / MAPK pathway may comprise a protein, peptide or small molecule that is capable of binding to the insulin receptor of the cell and activating the ERK / MAPK pathway. The activator of the ERK / MAPK pathway may comprise an insulin receptor (IR) agonist, which is preferably selective for the insulin receptor. For example, IR agonist peptides are known, such as S597

[0069] (SLEEEWAQIECEVYGRGCPSESFYDWFERQL) (SEQ ID NO: 6) or variants thereof, from Park et al. (Nat

[0070] Commun 13, 5594 (2022). which is herein incorporated by reference. The activator of the ERK / MAPK pathway may comprise a peptide that contains the insulin receptor binding sequence.

[0071] The activator of the ERK / MAPK pathway may comprise an insulin mimetic such as selected from the group consisting of exenatide (Byetta, Bydureon), liraglutide (Victoza), sitagliptin (Januvia, Janumet,

[0072] Janumet XR, Juvisync), saxagliptin (Onglyza, Kombiglyze XR), alogliptin (Nesina, Kazano, Oseni), and

[0073] I inagliptin (Tradjenta, Jentadueto). The activator of the ERK / MAPK pathway may comprise an insulin mimetic such as selenium.

[0074] The insulin may be natural or synthetic. The insulin of the composition may comprise human, bovine, or porcine insulin. Preferably, the insulin of the composition comprises human insulin. The insulin of the composition may comprise a polypeptide according to the sequence of SEQ ID NO: 1, a polypeptide according to the sequence of SEQ ID NO: 2, or a truncation of either, or a sequence that has at least

[0075] 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to either SEQ ID NO: 1-2.

[0076] FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 1) (B Chain)

[0077] GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 2) (A Chain)

[0078] The insulin of the composition may comprise bovine insulin. The insulin of the composition may comprise a polypeptide according to the sequence of SEQ ID NO: 3, a polypeptide according to the sequence of SEQ ID NO: 4, or a truncation of either, or a sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to either SEQ ID NO: 3-4.

[0079] FVNQHLCGSHLVEALYLVCGERGFFYTPKA (SEQ ID NO: 3)

[0080] GIVEQCCASVCSLYQLENYCN (DEQ ID NO: 4)

[0081] The insulin of the composition may comprise porcine insulin. The insulin of the composition may comprise a polypeptide according to the sequence of SEQ ID NO: 2, and / or a polypeptide according to the sequence of SEQ. ID NO: 3, or a truncation of either, or a sequence that has at least 70%, at least

[0082] 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to either SEQ ID NO: 2-3.

[0083] Alternatively or additionally, the insulin of the composition may comprise an insulin whose polypeptide sequence is derived from a non-human insulin orthologue, for example by amino acid substitution, or by sequence truncation, while retaining all, or substantial, function. The insulin may be recombinant, or may be purified from an animal. The insulin may have a molecular weight of about 5,808.0 Da. The person skilled in the art will be familiar with commercial sources of insulin.

[0084] The insulin may initially be provided in the composition as a powder to be dissolved therein.

[0085] Alternatively, the insulin may be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated insulin solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline (PBS) or Dulbecco's PBS (DPBS), or Earle's Balanced

[0086] Salt Solution (EBSS) without Phenol Red. Preferably, the concentrated insulin solution is in EBSS without Phenol Red.

[0087] Preferably, the insulin is provided in the composition at a concentration of about 20 mg / L, which is about 3.442 μM. The insulin may be provided in the composition at a concentration from about 2 mg / L to about 50 mg / L, which may be from about 0.3442 μM to about 8.605 μM. The insulin may be provided in the composition at a concentration of at least about 2 mg / L, at least about 5 mg / L, at least about 10 mg / L, at least about 15 mg / mL, at least about 17 mg / L, at least about 18 mg / L, at least about 19 mg / L, at least about 20 mg / L, at least about 21 mg / L, at least about 22 mg / L, at least about

[0088] 23 mg / L, at least about 24 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 40 mg / L, or at least about 50 mg / L. The iron carrier

[0089] The iron carrier may comprise transferrin. The transferrin of the composition may comprise human transferrin. The insulin of the composition may comprise a polypeptide according to the sequence of

[0090] SEQ ID NO: 5, or a truncation thereof, or a sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to SEQ ID NO: 5.

[0091] MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEAD

[0092] AVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGL

[0093] LYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFE

[0094] NLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSS

[0095] PHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNS

[0096] VGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAVAVVKKSASDL

[0097] TWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGY

[0098] YGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVT

[0099] RKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCS

[0100] TSSLLEACTFRRP (SEQ ID NO: 5)

[0101] Alternatively or additionally, the transferrin of the composition may comprise a transferrin whose polypeptide sequence is derived from a non-human insulin orthologue, for example by amino acid substitution, or by sequence truncation, while retaining all, or substantial, function. The transferrin may be recombinant, or may be purified from an animal. The transferrin may have a molecular weight of about 77,050 Da, or about 80,000 Da. The person skilled in the art will be familiar with commercial sources of transferrin. Transferrin may be provided bound to iron cations, and / or bound to other metal cations, or alternatively as an apoprotein.

[0102] The transferrin may initially be provided in the composition as a powder to be dissolved therein.

[0103] Alternatively, the transferrin may be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated transferrin solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline (PBS) or Dulbecco's PBS (DPBS), or Earle's

[0104] Balanced Salt Solution (EBSS) without Phenol Red. Preferably, the concentrated transferrin solution is in EBSS without Phenol Red. Preferably, the transferrin is provided in the composition at a concentration of about 11 mg / L, which is about 0.1375 μM. The transferrin may be provided in the composition at a concentration from about

[0105] 1 mg / L to about 30 mg / L, which may be from about 0.0125 μM to about 0.375 μM. The insulin may be provided in the composition at a concentration of at least about 1 mg / L, at least about 5 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about

[0106] 10 mg / L, at least about 11 mg / L, at least about 12 mg / L, at least about 13 mg / L, at least about 14 mg / L, at least about 15 mg / L, at least about 20 mg / L, or at least about 30 mg / L.

[0107] The Selenium Supplement

[0108] The selenium supplement is in the composition in addition to any selenium that may be comprised in the basal medium, such as IMDM. The selenium supplement may comprise sodium selenite, potassium selenite, lithium selenite, or other selenium salts. In a preferred embodiment, the selenium supplement comprises sodium selenite.

[0109] The selenium supplement may initially be provided in the composition as a powder to be dissolved therein. Alternatively, the selenium supplement may be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated selenium supplement solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline (PBS) or Dulbecco's PBS (DPBS), or Earle's Balanced Salt Solution (EBSS) without Phenol Red. Preferably, the concentrated selenium supplement solution is in EBSS without Phenol Red.

[0110] Preferably, the selenium supplement is provided in the composition at a concentration of about

[0111] 0.07746 μM. The selenium supplement may be provided in the composition at a concentration of from about 0.03 μM to about 0.15 μM. The selenium supplement may be provided in the composition at a concentration of at least about 0.03 μM, at least about 0.04 μM, at least about 0.05 μM, at least about

[0112] 0.055 μM, at least about 0.06 μM, at least about 0.065 μM, at least about 0.067 μM, at least about

[0113] 0.069 μM, at least about 0.070 μM, at least about 0.071 μM, at least about 0.072 μM, at least about

[0114] 0.073 μM, at least about 0.074 μM, at least about 0.075 μM, at least about 0.076 μM, at least about

[0115] 0.078 μM, at least about 0.079 μM, at least about 0.080 μM, at least about 0.081 μM, at least about

[0116] 0.082 μM, at least about 0.083 μM, at least about 0.085 μM, at least about 0.090 μM, at least about

[0117] 0.095 μM, at least about 0.10 μM, at least about 0.11 μM, at least about 0.12 μM, or at least about 0.15 μM. These concentration values do not include (and, therefore, are in addition to) the concentration of selenium, or selenium compound(s), that is also in the basal medium, such as IMDM.

[0118] The Cell Growth and Proliferation Promoter

[0119] The cell growth and proliferation promoter may be a small molecule (e.g. less than lkDa), such as an amino alcohol. The cell growth and proliferation promoter may comprise one of the agents selected from ethanolamine. carbinoxamine, clemastine. dimenhydrinate. chlorphenoxamine. diphenhydramine and doxylamine, or combinations thereof. A class of antihistamines is identified as ethanolamines, which includes carbinoxamine, clemastine, dimenhydrinate, chlorphenoxamine, diphenhydramine and doxylamine, and which may be the cell growth and proliferation promoter. In one embodiment the cell growth and proliferation promoter comprises ethanolamine. The ethanolamine may be 2-aminoethanol. The ethanolamine may initially be provided in the composition as a powder to be dissolved therein. Alternatively, the ethanolamine may be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated ethanolamine solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline

[0120] (PBS) or Dulbecco's PBS (DPBS), or Earle's Balanced Salt Solution (EBSS) without Phenol Red. Preferably, the concentrated ethanolamine solution is in EBSS without Phenol Red.

[0121] Preferably, the ethanolamine is provided in the composition at a concentration of about 4.0 mg / L, which is about 0.06557 mM. The ethanolamine may be provided in the composition at a concentration from about 1 mg / L to about 10 mg / L, which is from about 0.8197 mM to about 8.1967 mM. The ethanolamine may be provided in the composition at a concentration of at least about 1 mg / L, at least about 1.5 mg / L, at least about 2 mg / L, at least about 3 mg / L, at least about 3.5 mg / L, at least about

[0122] 3.6 mg / L, at least about 3.7 mg / L, at least about 3.8 mg / L, at least about 3.9 mg / L, at least about 4.0 mg / L, at least about 4.1 mg / L, at least about 4.2 mg / L, at least about 4.3 mg / L, at least about 4.4 mg / L, at least about 4.5 mg / L, at least about 5 mg / L, at least about 6 mg / L, at least about 8 mg / L, at least about 9 mg / L, or at least about 10 mg / L. The concentrations provided for ethanolamine may be equally applicable for the concentrations of any cell growth and proliferation promoter, such as those described herein.

[0123] The first anti-oxidant The first anti-oxidant may further promote cell proliferation. The anti-oxidant may be a small molecule

[0124] (e.g. less than lkDa). The first anti-oxidant may comprise vitamin A, vitamin C, vitamin E, monothioglycerol or 2-mercaptoethanol, or any combination thereof. The first anti-oxidant may comprise monothioglycerol or 2-mercaptoethanol. In one embodiment, the first anti-oxidant comprises monothioglycerol.

[0125] The monothioglycerol (or thioglycerol, or 1 -thioglycerol) may comprise mono-thio glycerol. The mono- thio glycerol may initially be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated mono-thio glycerol solution may be e.g. an aqueous solution, or a solution of basal medium, such as phosphate-buffered saline (PBS) or

[0126] Dulbecco's PBS (DPBS) or IMDM. Preferably, the concentrated mono-thio glycerol solution is in the basal medium, such as IMDM, according to the invention.

[0127] Preferably, the first anti-oxidant, such as mono-thio glycerol, is provided in the composition at a concentration of about 0.004% (v / v). The first anti-oxidant, such as mono-thio glycerol, may be provided in the composition at a concentration from about 0.0020% (v / v) to about 0.010% (v / v). The first anti-oxidant, such as mono-thio glycerol, may be provided in the composition at a concentration of at least about 0.0020% (v / v), at least about 0.0025% (v / v), at least about 0.0030% (v / v), at least about 0.0035% (v / v), at least about 0.0036% (v / v), at least about 0.0037% (v / v), at least about 0.0038%

[0128] (v / v), at least about 0.0039% (v / v), at least about 0.0040% (v / v), at least about 0.0041% (v / v), at least about 0.0042% (v / v), at least about 0.0043% (v / v), at least about 0.0044% (v / v), at least about 0.0045%

[0129] (v / v), at least about 0.0050% (v / v), at least about 0.0060% (v / v), at least about 0.0070% (v / v), at least about 0.0080% (v / v), at least about 0.0090% (v / v), or at least about 0.010% (v / v).

[0130] The second anti-oxidant

[0131] The second anti-oxidant may also promote cell survival and proliferation. The second antioxidant may comprise ascorbic acid-2-phosphate (AA2P). The second antioxidant, such as AA2P, may initially be provided in the composition as a powder to be dissolved therein. Alternatively, the second antioxidant, such as AA2P, may be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated anti-oxidant solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline (PBS) or Dulbecco's PBS (DPBS). Preferably, the second antioxidant, such as AA2P, is provided in the composition at a concentration of about 64.0 mg / L. The second antioxidant, such as AA2P, may be provided in the composition at a concentration from about 30.0 mg / L to about 100 mg / L. The second antioxidant, such as AA2P, may be provided in the composition at a concentration of at least about 30.0 mg / L, at least about 40.0 mg / L, at least about 50.0 mg / L, at least about 55.0 mg / L, at least about 59.0 mg / L, at least about 60.0 mg / L, at least about 61.0 mg / L, at least about 62.0 mg / L, at least about 63.0 mg / L, at least about 64.0 mg / L, at least about 65.0 mg / L, at least about 66.0 mg / L, at least about 67.0 mg / L, at least about 68.0 mg / L, at least about 69.0 mg / L, at least about 70.0 mg / L, at least about 71.0 mg / L, at least about 75.0 mg / L, at least about 80.0 mg / L, at least about 90.0 mg / L, or at least about 100 mg / L.

[0132] The first and second antioxidants may be different to each other.

[0133] The L-Glutamine Supplement

[0134] The L-glutamine supplement may comprise L-alanyl-L-glutamine. The L-glutamine supplement may initially be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated L-glutamine supplement solution may be e.g. an aqueous solution, or a 0.85% (w / v) NaCI aqueous solution, or an aqueous solution, or a solution of phosphate- buffered saline (PBS) or Dulbecco's PBS (DPBS). Preferably, the concentrated L-glutamine supplement solution is a 0.85% (w / v) NaCI aqueous solution.

[0135] Preferably, the L-glutamine supplement is provided in the composition at a concentration of about 2.0 mM. The L-glutamine supplement may be provided in the composition at a concentration from about

[0136] 0.5 mM to about 10.0 mM. The L-glutamine supplement may be provided in the composition at a concentration of at least about 0.5 mM, at least about 0.6 mM, at least about 0.7 mM, at least about

[0137] 1.0 mM, at least about 1.5 mM, at least about 1.6 mM, at least about 1.7 mM, at least about 1.8 mM, at least about 1.9 mM, at least about 2.0 mM, at least about 2.1 mM, at least about 2.2 mM, at least about 2.3 mM, at least about 2.4 mM, at least about 2.5 mM, at least about 3.0 mM, at least about

[0138] 4.0 mM, at least about 5.0 mM, at least about 7.0 mM, or at least about 10.0 mM.

[0139] The Non-Essential Amino Acids Supplement

[0140] The non-essential amino acids (NEAA) supplement may comprise or consist of one or more of glycine,

[0141] L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. The NEAA supplement is in the composition in addition to any amino acids that may be comprised in any of the other composition components. The components of the NEAA supplement may initially be provided in the composition as a powder to be dissolved therein. Alternatively, the NEAA supplement may be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated NEAA supplement solution may be e.g. an aqueous solution, or a solution of phosphate- buffered saline (PBS) or Dulbecco's PBS (DPBS). Preferably, the concentrated NEAA supplement solution is an aqueous solution.

[0142] Preferably, the NEAA supplement is provided in the composition at a concentration of about 100 μM.

[0143] This concentration refers to the concentration of each component of the NEAA supplement, i.e. in embodiments where the NEAA supplement comprises the seven amino acids of the previous paragraph, each of said amino acids is preferably present in the composition at a concentration of 100 μM. The NEAA supplement may be provided in the composition at a concentration from about 20 μM to about 1 mM. The NEAA supplement may be provided in the composition at a concentration of at least about 20 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about

[0144] 80 μM, at least about 90 μM, at least about 92 μM, at least about 95 μMat least, about 97 μM, at least about 100 μM, at least about 102 μM, at least about 105 μM, at least about 107 μM, at least about 110 μM, at least about 120 μM, at least about 130 μM, at least about 150 μM, at least about

[0145] 200 μM, at least about 300 μM, at least about 500 μM, at least about 700 μM, or at least about 1 mM.

[0146] These concentration values do not include (and, therefore, are in addition to) the concentration of any amino acids that are also in any of the other composition components.

[0147] The antibiotic

[0148] The antibiotic may comprise a penicillin and / or streptomycin, and / or functional derivatives thereof.

[0149] The antibiotic may initially be provided in the composition as a powder to be dissolved therein.

[0150] Alternatively, the antibiotic may be provided as a solution, e.g. a concentrated solution that is diluted in the composition. In such embodiments, the concentrated antibiotic solution may be e.g. an aqueous solution, or a solution of phosphate-buffered saline (PBS) or Dulbecco's PBS (DPBS). Preferably, the concentrated antibiotic supplement solution is an aqueous solution.

[0151] In a preferred embodiment, the antibiotic comprises penicillin and streptomycin. In some embodiments, the penicillin is supplied in the composition at a concentration of about 100 units / mL and the streptomycin at a concentration of about 0.1 mg / mL. Alternatively, in these embodiments, the penicillin may be supplied in the composition at a concentration of from about 10 units / mL to about

[0152] 200 units / mL, and the streptomycin at a concentration of from about 0.05 mg / mL to about 2 mg / mL.

[0153] The penicillin may be supplied in the composition at a concentration of at least about 10 units / mL, at least about 20 units / mL, at least about 30 units / mL, at least about 40 units / mL, at least about 50 units / mL, at least about 60 units / mL, at least about 70 units / mL, at least about 80 units / mL, at least about 90 units / mL, or at least about 100 units / mL. The streptomycin may be supplied in the composition at a concentration of at least about 0.05 mg / mL, at least about 0.08 mg / mL, at least about

[0154] 0.09 mg / mL, or at least about 0.1 mg / mL.

[0155] The chemically defined universal medium (CDUM)

[0156] Preferably the chemically defined medium (CDUM) does not comprise albumin, such as bovine albumin.

[0157] According to one aspect, the CDUM medium comprises from about 50% (v / v) to about 80% (v / v) basal medium, such as IMDM, that comprises Phenol Red (for example, IMDM of composition according to

[0158] Table 2), from about 15% (v / v) to about 30% (v / v) L-alanyl-L-glutamine-free second basal medium (for example, F12 of the CDUM medium according to Table 3), from about 5 mg / mL to about 15 mg / mL polymer, such as PVA, from about 5 mg / L to about 30 mg / L lipids, from about 0.3442 μM to about

[0159] 8.605 μM activator of the ERK / MAPK pathway, such as insulin, from about 0.0125 μM to about 0.375 μM iron carrier, such as transferrin, from about 0.030 μM to about 0.150 μM selenium supplement, from about 0.8197 mM to about 8.1967 mM proliferation promoter, such as ethanolamine, from about

[0160] 0.0020% (v / v) to about 0.010% (v / v) antioxidant, such as mono-thio glycerol, from about 30.0 mg / L to about 100 mg / L second antioxidant, such as AA2P, from about 0.5 mM to about 10.0 mM L-glutamine supplement, from about 20 μM to about 1 mM μM NEAA supplement and, optionally, from about 10 units / mL to about 200 units / mL penicillin and from about 10 pg / mL to about 200 pg / mL streptomycin.

[0161] This preferred aspect may be referred to as chemically defined endoderm medium (CDEM). According to one preferred aspect, the CDUM medium comprises about 70.32% (v / v) basal medium, such as

[0162] IMDM that comprises Phenol Red (for example, IMDM of composition according to Table 2), about

[0163] 23.44% (v / v) L-alanyl-L-glutamine-free second basal medium (for example, F12 of composition according to Table 3), about 10 mg / mL polymer, such as PVA, about 8 mg / L lipids, about 3.442 μM activator of the ERK / MAPK pathway, such as insulin, about 0.1375 μM iron carrier, such as transferrin. about 0.07746 μM selenium supplement, about 0.06557 mM proliferation promoter, such as ethanolamine, about 0.0039% (v / v) antioxidant, such as mono-thio glycerol, about 64.0 mg / L second antioxidant, such as AA2P, about 2.0 mM L-glutamine supplement, about 100 μM NEAA supplement and, optionally, about 100 units / mL penicillin and about O.lmg / mL streptomycin. This preferred aspect may also be referred to as CDEM.

[0164] Chemically defined endoderm medium (CDEM) may be considered as a specific form of CDUM, wherein the ratio of the first basal medium to the second basal media is greater. CDUM, may comprise a ratio of the first basal medium to the second basal media of about 0.5-2.4:!. In one embodiment, the CDUM, may comprise a ratio of the first basal medium to the second basal media of about 1:1.

[0165] CDEM may be a type of CDUM that comprises a ratio of the first basal medium to the second basal media of about 2.5-4:l or greater than 2.5:1. In one embodiment, the CDEM may be a type of CDUM that comprises a ratio of the first basal medium to the second basal media of about 3:1, or greater than 3:1, such as 3-5:1. In one embodiment, the CDEM may be a type of CDUM that comprises a ratio of the first basal medium to the second basal media of about 3:1.

[0166] The total basal medium (first and second) in CDUM may be at least 65% v / v. In another embodiment, the total basal medium (first and second) in CDUM may be at least 80% v / v. In another embodiment, the total basal medium (firstand second) in CDUM may be at least 90% v / v. In a preferred embodiment. the total basal medium (first and second) in CDUM may be about 94% v / v, or at least 94% v / v.

[0167] According to another aspect, the CDUM medium further comprises from about 35% (v / v) to about

[0168] 60% (v / v) Phenol Red-free basal medium, such as IMDM (for example, IMDM of composition according to Table 1), from about 35% (v / v) to about 60% (v / v) nutrient mixture, such as F12, that comprises L- alanyl-L-glutamine (for example, F12 of the CDUM medium according to Table 4), from about 5 mg / mL to about 15 mg / mL polymer, such as PVA, from about 5 mg / L to about 30 mg / L lipids, from about

[0169] 0.3442 μM to about 8.605 μM activator of the ERK / MAPK pathway, such as insulin, from about 0.0125 μM to about 0.375 μM iron carrier, such as transferrin, from about 0.030 μM to about 0.150 μM selenium supplement, from about 0.8197 mM to about 8.1967 mM proliferation promoter, such as ethanolamine, from about 0.0020% (v / v) to about 0.010% (v / v) antioxidant, such as mono-thio glycerol, from about 30.0 mg / L to about 100 mg / L second antioxidant, such as AA2P, from about 0.5 mM to about 10.0 mM L-glutamine supplement, from about 20 μM to about 1 mM μM NEAA supplement and, optionally, from about 5 units / mL to about 100 units / mL penicillin and from about 5 pg / mL to about 200 pg / mL streptomycin. This preferred aspect may be referred to as chemically defined universal medium (CDUM). According to one preferred aspect, the composition comprises about 46.9% (v / v) Phenol Red-free basal medium, such as IMDM (for example, IMDM of composition according to Table 1), about 46.9% (v / v) nutrient mixture, such as F12, that comprises L-alanyl-L glutamine (for example, F12 of composition according to Table 4), about 10 mg / mL polymer, such as

[0170] PVA, about 8 mg / L lipids, about 3.442 μM activator of the ERK / MAPK pathway, such as insulin, about

[0171] 0.1375 μM iron carrier, such as transferrin, about 0.07746 μM selenium supplement, about 0.06557 mM proliferation promoter such as ethanolamine, about 0.0039% (v / v) antioxidant such as mono-thio glycerol, about 64.0 mg / L second antioxidant such as AA2P, about 2.0 mM L-glutamine supplement, about 100 μM NEAA supplement and, optionally, about 100 units / mL penicillin and about O.lmg / mL streptomycin. This preferred aspect may also be referred to as CDUM.

[0172] The components of the composition may be sterile when provided in the composition, for example they may be rendered sterile by filtration through a 0.22-pm filter. Alternatively or additionally, the composition may be rendered sterile, such as by filtration, once at least two of its components have been mixed.

[0173] According to another aspect, there is provided a composition according to the current invention that comprises no components derived from species other than human. Such derivation refers to inclusion in the composition by way of isolation / purification, and inclusion by way of the composition comprising sequences that are non-human.

[0174] According to another aspect, there is provided a composition substantially according to the current invention that comprises no serum and also no serum-derived products.

[0175] The composition, either alone or suitably supplemented with further components, may allow the maintenance of hiPSCs in culture for a period of at least about 6 hours, or least about 12 hours, or least about 18 hours, or least about 24 hours, or least about 30 hours, or least about 36 hours, or least about 42 hours, or least about 48 hours, or least about 54 hours, or least about 60 hours, or least about

[0176] 72 hours, or least about 84 hours, or least about 96 hours, or least about 108 hours, or least about 120 hours, or least about 132 hours.

[0177] The composition, either alone or suitably supplemented with further components, may allow the induction of, or preparation for, differentiation of the hiPSCs towards selected cell lineages in culture over a period of at least about 6 hours, or least about 12 hours, or least about 18 hours, or least about

[0178] 24 hours, or least about 30 hours, or least about 36 hours, or least about 42 hours, or least about 48 hours, or least about 54 hours, or least about 60 hours, or least about 72 hours, or least about 84 hours, or least about 96 hours, or least about 108 hours, or least about 120 hours, or least about 132 hours.

[0179] The hiPSCs

[0180] The hiPSCs may be derived from somatic cells, such as fibroblasts, CD34+cells, or PBMCs (peripheral blood mononuclear cells). The hiPSCs may be derived from cancer cells. The hiPSCs may be derived from one lineage of adult somatic cells, such as fibroblasts. Alternatively or additionally, the hiPSCs may be derived from lineages of immature of non-terminally differentiated cells, such as CD34+cells or PBMCs. Alternatively, the hiPSCs may be derived from a mixture of cells of multiple sources and / or lineages. Preferably, the hiPSCs are derived from cells of mesodermal lineages. Alternatively, the hiPSCs may be derived from cells of other germ layer lineages, i.e. endodermal and ectodermal lineages. Alternatively, the original germ layer lineage of the source or sources of hiPSCs may be undetermined or unclear.

[0181] The hiPSCs may be derived from somatic adult, or progenitor, cells obtained from more than one human or non-human animal which have been subsequently pooled.

[0182] The person skilled in the art will be familiar with a variety of methods for obtaining hiPSCs from somatic cell lineages, and will employ hiPSCs that have been derived via the method or methods that are most suited to the various embodiments of the invention described herein. For example, in some embodiments, it may be appropriate to use a chemical (and / or temperature-based) induction method for hiPSC derivation. In other embodiments, it may be appropriate to use a vector-based induction method for hiPSC derivation. In some preferred embodiments, hiPSCs are derived from somatic cells via a viral infection method. Similarly, a particular source of starting cells, e.g. CD34+cells, may be most appropriate for a particular embodiment as described herein, and the person skilled in the art will be able to select hiPSCs that have been derived from the most suitable type of somatic cells.

[0183] It may be advantageous for the hiPSC derivation method not to make use of, or comprise, any other

[0184] ("feeder") cells in co-culture as a source of growth factors and / or source of conditioned growth medium. In preferred embodiments, hiPSCs are derived via a feeder-free culturing protocol. Alternatively, hiPSCs may be derived via feeder-based culturing methods. Feeder-based culturing methods may comprise cell types that confer growth and pluripotent characteristics to cells. Feeder-based culturing methods may comprise dermal fibroblast cells such as primary human dermal fibroblasts, or embryonic fibroblast cells such as primary mouse embryonic fibroblasts, as the feeder cells. It may be advantageous for the hiPSC derivation method not to employ non-human-animal- derived products, such as blood serum, or extracellular-membrane glycoprotein preparations. In some embodiments, the hiPSC derivation method employs recombinant laminin, such as laminin-11 or laminin-121, as the physical substrate onto which the hiPSC being derived are cultured at least temporarily. Alternatively or additionally, the hiPSCs are derived via a method that employs ECM, such as Matrigel™, as the physical substrate for culture at least temporarily. hiPSCs that are cultured with the composition according to the present invention may be differentiated into other types of cells. In embodiments, the hiPSCs may be cultured in growth medium that is different to the composition of the current invention for a length of time beforehand and / or afterwards. In some embodiments, the hiPSCs may be cultured in a maintenance medium, and / or in the presence of feeder cells, before they are cultured in the composition. In some embodiments, the hiPSCs may be cultured in growth medium that comprises the composition of the current invention and other components, referred to as agents. In particular, the hiPSCs may be cultured in the composition that has been further treated by addition of agents such as growth factors, cytokines, chemokines, polypeptides, lipids, sugars, and / or a mixture thereof. The addition of specific agents to the composition may allow the induction of differentiation of the hiPSCs towards selected cell lineages in culture.

[0185] The hiPSC Maintenance Medium

[0186] The hiPSC maintenance medium may comprise any medium that is suitable to the indefinite, or long- term, or medium-term, or short-term, culturing of pluripotent stem cells. The hiPSC maintenance medium may comprise medium that is commonly referred to in the art as MTeSR™ medium, or

[0187] MTeSR™ 1 medium, MTeSR™ Plus medium, E8 medium, or Essential 8™ Medium. The hiPSC maintenance medium may comprise a medium substantially produced according to the teaching of the journal article G. Chen G et AL, Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011 May;8(5):424-9, which is herein incorporated in its entirety by reference; this medium composition is referred to as E8 medium hereinafter. The person skilled in the art will be familiar with culturing protocols, such as cell passaging protocols, that may employ the hiPSC maintenance medium and that are appropriate to the maintenance of hiPSCs in culture.

[0188] Other Aspects

[0189] According to another aspect of the present invention, there is provided the chemically defined universal medium according to the invention, further comprising a differentiation factor selected from

[0190] BMP pathway activator, such as BMP4, an activator of SMAD2 / 3 signalling, such as Activin A, a Wnt signalling activator, such as CHIR99021, a PI3K inhibitor, such as LY294002, an activator of VEGF signalling, such as VEGF, an activator of FGF signalling, such as FGF2, an activator of PKA signalling, such as 8Bro-cAMP or cAMP, an activator of EGF signalling, such as EGF, an insulin receptor agonist, such as insulin, a P-adrenergic agonist, such as Isoprenaline, a corticosteroid, such as hydrocortisone. a TGF-Beta superfamily inhibitor, such as SB431542, a c-kit receptor (CD117) agonist, such as SCF, an

[0191] IL-3 receptor agonist, such as IL-3, an IL-4 receptor agonist, such as IL-4, an IL-6 receptor agonist and / or

[0192] GP130 agonist, such as IL-6, a TPO (thrombopoietin) receptor agonist, such as TPO, a c-Fms receptor agonist, such as M-CSF, GM-CSF receptor agonist, such as GM-CSF, an IFNAR agonist, such as IFN- gamma, an immunogen, such as LPS, a BMP pathway inhibitor, such as dorsomorphin, a Wnt signalling activator, such as Wnt3A, an agent to potentiate Wnt signalling, such as R-Spondinl, retinoic acid receptor agonist, such as retinoic acid, FGF receptor agonist, such as FGF10, FGFR2IIIB receptor agonist, such as FGF7, a phosphodiesterase inhibitor, such as IBMX, another activator of PKA signalling, such as cAMP, and another corticosteroid, such as DEXA or Hydrocortisone; or combinations thereof.

[0193] Mesoderm induction medium

[0194] According to another aspect of the present invention, there is provided a mesoderm induction medium, wherein the mesoderm induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a BMP pathway activator, such as BMP4, ii) an activator of SMAD2 / 3 signalling, such as Activin A, iii) a Wnt signalling activator, such as CHIR99021, and iv) a PI3K inhibitor, such as LY294002. Each of the components i)-iv) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0195] The BMP pathway activator may activate the BMP pathway via the BMP receptors and may activate

[0196] SMAD and MARK mediated intracellular signalling cascades to affect gene expression. The skilled person will recognise that BMP4 is one such BMP pathway activator, yet multiple other BMPs can activate similar signalling in cells that may either entirely or partially overlap with the effect on BMP4.

[0197] This may include any of the BMP family of proteins, e.g. BMP1-15. Any other BMP recombinant protein, either full length or partial that is able to bind the receptor could be used instead of BMP4.

[0198] Also, small molecule BMP pathway activators are available, which are both naturally occurring and synthesised may activate the BMP pathway, for example SB 4 (2-[[(4-

[0199] Bromophenyl)methyl]thio]benzoxazole) is known to activate canonical BMP signaling and increases

[0200] SMAD-1 / 5 / 9 phosphorylation.

[0201] The BMP pathway activator, such as BMP4, may be provided at a concentration of about 50 ng / ml. The

[0202] BMP pathway activator, such as BMP4, may be provided at a concentration of at least 1 picogram / ml.

[0203] In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of at least 40 ng / ml. The BMP pathway activator, such as BMP4, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of from about 40 ng / ml to about

[0204] 60 ng / ml.

[0205] An activator of SMAD2 / 3 signalling may be Activin A, or a molecule having equivalent activity. As for

[0206] BMP, Activin A is part of the transforming growth factor beta (TGF-0) superfamily. Any of the activins, or TGF-[3s may substitute for Activin A. Similarly, the GDF subfamily can also substitute Activin A. The intracellular signalling pathway of interest is SMAD2 / 3 so any other protein and / or small molecule activating SMAD2 / 3 signalling could substitute Activin A. An example small molecule that activates

[0207] SMAD2 / 3 signalling includes SRI-011381-hydrochloride: The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of about

[0208] 15 ng / ml. The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as activin

[0209] A, may be provided at a concentration of at least lOng / ml. The activator of SMAD2 / 3 signalling, such as activin A, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as, activin A may be provided at a concentration of from about 10 ng / ml to about 20 ng / ml. The activator of SMAD2 / 3 signalling, such as Activin A, may only be provided in day 1 (D1) of the culture, for example if the mesoderm induction medium is changed / refreshed after 12-24 hours, the activator of SMAD2 / 3 signalling, such as Activin

[0210] A, may not be provided in the new mesoderm induction medium.

[0211] The Wnt signalling activator may be a small molecule inhibitor (i..e. <900Da) of the inhibitor of Wnt signalling (GSK3b), such as CHIR99021. The Wnt signalling activator may comprise IWR-1 (4-

[0212] (l,3,3a,4,7,7a-Hexahydro-l,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide):

[0213] In another embodiment, the Wnt signalling activator may be a Wnt family member capable of Wnt signalling activation. In one embodiment, the Wnt signalling activator may be a peptide activator, such as Wnt3a, or a functionally equivalent peptide thereof having the function of a 0-catenin pathway agonist.

[0214] In one embodiment, the Wnt signalling activator is CHIR99021. CHIR99021 may also be known as 6-

[0215] ((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-lH-imidazol-2-yl)pyrimidin-2- yl)amino)ethyl)amino)nicotinonitrile. The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of about 5 μM. The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of at least 1 μM. In another embodiment, the Wnt signalling activator, such as CHIR99021, may be provided at a concentration of at least 3 μM. The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the Wnt signalling activator, such as CHIR99021, may be provided at a concentration of from about 3 μM to about 8 μM. The Wnt signalling activator, such asCHIR99021, may only be provided in day 1 (DI) of the culture, for example if the mesoderm induction medium is changed / refreshed after

[0216] 12-24 hours, the Wnt signalling activator, such as CHIR99021, may not be provided in the new mesoderm induction medium.

[0217] In one embodiment, the PI3K inhibitor is LY294002:

[0218] A.

[0219] The skilled person will recognise that LY294002 is one example of a PI3K inhibitor, and other alternative

[0220] PI3K inhibitors may be provided, such as those selected from Dactolisib (BEZ235), Pictilisib (GDC-0941),

[0221] LY294002, Buparlisib (BKM120), PI-103, NU7441 (KU-57788), TGX-221, and IC-87114, or combinations thereof.

[0222] Dactolisib (BEZ235) may have the structure:

[0223] Pictilisib (GDC-0941) may have the structure: Buparlisib (BKM120) may have the structure:

[0224] PI-103 may have the structure:

[0225] NU7441 (KU-57788) may have the structure:

[0226] TGX-221 may have the structure:

[0227] /

[0228] IC-87114 may have the structure: The PI3K inhibitor, such as LY294002, may be provided at a concentration of about 10 μM. The PI3K inhibitor, such as LY294002, may be provided at a concentration of at least about 1 μM. In another embodiment, the PI3K inhibitor, such as LY294002, may be provided at a concentration of at least about 5 μM. The PI3K inhibitor, such as LY294002, may be provided at a concentration of from about

[0229] 1 μM to about 1 M. In another embodiment, the PI3K inhibitor, such as LY294002, may be provided at a concentration of from about 5 μM to about 15 μM.

[0230] Endothelial cell induction medium

[0231] According to another aspect of the present invention, there is provided an endothelial cell induction medium, wherein the endothelial cell induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) an activator of VEGF signalling, such as VEGF, ii) an activator of FGF signalling, such as FGF2, and iii) an activator of PKA signalling, such as cAMP or 8Bro-cAMP.

[0232] Each of the components i), ii), or iii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0233] The activator of VEGF signalling may comprise Gremlin, VEGF, VEGF mimetic peptides, such as

[0234] KLTWQELYQLKYKGI, or small molecule VEGF signalling activators, such as Taurocholic acid sodium or

[0235] Taurocholic acid-13Cj,15N (sodium).

[0236] The activator of VEGF signalling, such as VEGF, may be provided at a concentration of about 300 ng / ml.

[0237] The activator of VEGF signalling, such as VEGF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of VEGF signalling, such as VEGF, may be provided at a concentration of at least 200 ng / ml. The activator of VEGF signalling, such as VEGF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of VEGF signalling, such as VEGF, may be provided at a concentration of from about 200 ng / ml to about 400 ng / ml. The activator of FGF signalling may act via MAPK, ERK signalling. The skilled person will recognise that any of the FGF family members may be used. Alternatively, the activator of FGF signalling may comprise an antibody such as Fazpilodemab (BFKB8488A).

[0238] The activator of FGF signalling, such as FGF2, may be provided at a concentration of about 200 ng / ml.

[0239] The activator of FGF signalling, such as FGF2, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of FGF signalling, such as FGF2, may be provided at a concentration of at least 100 ng / ml. activator of FGF signalling, such as FGF2, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. in another embodiment, the activator of FGF signalling, such as FGF2, may be provided at a concentration of from about 100 ng / ml to about

[0240] 300 ng / ml.

[0241] The activator of PKA signalling may comprise one or more of 8Bro-cAMP: cAMPS-Sp:

[0242] CW 008: or cyclic adenosine monophosphate, also known as cyclic AMP or cAMP.

[0243] The activator of PKA signalling may induce VEGF production. The activator of PKA signalling, such as

[0244] 8Bro-cAMP, may be provided at a concentration of about 1 mM. The activator of PKA signalling, such as 8Bro-cAMP, may be provided at a concentration of at least about 1 μM. In another embodiment. the activator of PKA signalling, such as 8Bro-cAMP, may be provided at a concentration of at least about

[0245] 0.5 mM. The activator of PKA signalling, such as 8Bro-cAMP, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the activator of PKA signalling, such as 8Bro- cAMP, may be provided at a concentration of from about 0.5 mM to about 1.5 mM.

[0246] Fibroblast induction medium

[0247] According to another aspect of the present invention, there is provided a fibroblast induction medium, wherein the fibroblast induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) an activator of EGF signalling, such as EGF, ii) an insulin receptor agonist, such as insulin, iii) a BMP pathway activator, such as BMP4, iv) a P-adrenergic agonist, such as isoprenaline, and v) a corticosteroid, such as hydrocortisone.

[0248] Each of the components i)-v) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0249] The activator of EGF signalling may comprise an agent, such as a peptide, polypeptide or small molecule that activates intracellular signalling via GRB, SOS, PCK, NFKB, and / or PI3K. The activator of

[0250] EGF signalling may comprise one or more of EGF, amphiregulin, TGF-a, and heregulin. The activator of EGF signalling, such as EGF, may be provided at a concentration of about 10 ng / ml.

[0251] The activator of EGF signalling, such as EGF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of EGF signalling, such as EGF, may be provided at a concentration of at least 5 ng / ml. The activator of EGF signalling, such as EGF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, theactivator of

[0252] EGF signalling, such as EGF, may be provided at a concentration of from about 5 ng / ml to about 20 ng / ml.

[0253] The insulin receptor agonist may activate signalling via the insulin receptor and intracellular signalling via mapk, pi3k, akt, gsk3b and nfKb. The insulin receptor agonist may comprise a natural or synthetic full or partial length insulin analogue protein or peptide. The insulin receptor agonist may comprise one or more of insulin, GIP, GIP (1-30) amide, [D-Ala2]-GIP, (Pro3) GIP, GIP (l-30)-Myr, BCTC, azemiglitazone potassium, insulin efsitora alfa, GLP-1R / GIPR agonist-1, DA-JC4, 01338 and PTP1B-IN-

[0254] 24.

[0255] The BCTC structure may be: ci

[0256] The azemiglitazone potassium structure may be:

[0257] The PTP1B-IN-24 structure may be: 6 o

[0258] The insulin receptor agonist, such as insulin, may be provided at a concentration of about 5 μg / ml. The insulin receptor agonist, such as insulin, may be provided at a concentration of at least 1 picogram / ml.

[0259] In another embodiment, the insulin receptor agonist, such as insulin, may be provided at a concentration of at least 3 μg / ml. The insulin receptor agonist, such as insulin, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the insulin receptor agonist, such as insulin, may be provided at a concentration of from about 3 μg / ml to about

[0260] 10 μg / ml.

[0261] The BMP pathway activator may be as described herein above. The BMP pathway activator, such as

[0262] BMP4, may be provided at a concentration of about 12.8ng / ml. The BMP pathway activator, such as

[0263] BMP4, may be provided at a concentration of at least about 1 picogram / ml. In another embodiment. the BMP pathway activator, such as BMP4, may be provided at a concentration of at least about 1 ng / ml. The BMP pathway activator, such as BMP4, may be provided at a concentration of from about

[0264] 1 picogram / ml to about 1 g / ml. In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of from about 5 ng / ml to about 20ng / ml.

[0265] The p-adrenergic agonist may be selected from the group comprising (R)-adrenaline, l-(4- methoxyphenyl)-2-{[4-(4-nitrophenyl)butan-2-yl]amino}ethanol, L-isoprenaline, abediterol, albuterol,

[0266] Anoro Ellipta, apraclonidine, arbutamine, arformoterol, arformoterol fumarate, bambuterol, bambuterol hydrochloride, bitolterol, bitolterol mesylate, Breo Ellipta, butopamine, clenbuterol, clenbuterol hydrochloride, clorprenaline, colterol, diazoxide, dobutamine, dobutamine hydrochloride, dopamine, fenoterol, fenoterol hydrobromide, formoterol, formoterol fumarate, indacaterol, indacaterol maleate, isoprenaline, methoxyphenamine, mirabegron, olodaterol, olodaterol hydrochloride, orciprenaline, ractopamine, salmeterol, solabegron, terbutaline, vibegron, vilanterol, and vilanterol trifenate; or combinations thereof.

[0267] In one embodiment, the p-adrenergic agonist comprises isoprenaline. The p-adrenergic agonist, such as isoprenaline, may be provided at a concentration of about 2 μg / ml.

[0268] The p-adrenergic agonist, such as isoprenaline, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the P-adrenergic agonist, such as isoprenaline, may be provided at a concentration of at least 1 μg / ml. The P-adrenergic agonist, such as isoprenaline, may be provided at a concentration of from about 1 picogram / ml to about 3 g / ml. In another embodiment, the β- adrenergic agonist, such as isoprenaline, may be provided at a concentration of from about 1 μg / ml to about 3 μg / ml.

[0269] The corticosteroid may bind to the glucocorticoid receptor leading to downstream effects such as one or more of inhibition of phospholipase A2, NF-kappa B, other inflammatory transcription factors, and the promotion of anti-inflammatory genes. In one embodiment, the corticosteroid comprises hydrocortisone. The skilled person will recognise that there are several natural and synthetic corticosteroids that are capable of binding to the glucocorticoid receptor and substituting for hydrocortisone.

[0270] The corticosteroid, such as hydrocortisone, may be provided at a concentration of about 0.5 μg / ml.

[0271] The corticosteroid, such as hydrocortisone, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the corticosteroid, such as hydrocortisone, may be provided at a concentration of at least 0.2 μg / ml. The corticosteroid, such as hydrocortisone, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the corticosteroid, such as hydrocortisone, may be provided at a concentration of from about 0.2 μg / ml to about 1 μg / ml.

[0272] Hemogenic endothelium induction medium

[0273] According to another aspect of the present invention, there is provided a hemogenic endothelium induction medium, wherein the hemogenic endothelium induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) an activator of VEGF signalling, such as VEGF, ii) an activator of FGF signalling, such as FGF2, iii) a TGF-Beta superfamily inhibitor, such as SB431542, and iv) c-kit receptor (CD117) agonist, such as SCF. Each of the components i)-iv) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0274] The activator of VEGF signalling may be as described herein. The activator of VEGF signalling, such as

[0275] VEGF, may be provided at a concentration of about 50 ng / ml. The activator of VEGF signalling, such as

[0276] VEGF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of VEGF signalling, such as VEGF, may be provided at a concentration of at least 30 ng / ml.

[0277] The activator of VEGF signalling, such as VEGF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of VEGF signalling, such as VEGF, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0278] The activator of FGF signalling may be as described herein. The activator of FGF signalling, such as

[0279] FGF2, may be provided at a concentration of about 50 ng / ml. The activator of FGF signalling, such as

[0280] FGF2, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of FGF signalling, such as FGF2, may be provided at a concentration of at least 30 ng / ml. The activator of FGF signalling, such as FGF2, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of FGF signalling, such as FGF2, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0281] The TGF-Beta superfamily inhibitor may inhibit signalling via SMAD2 / 3. In one embodiment, the TGF-

[0282] Beta superfamily inhibitor comprises SB431542 (also known as 4-[4-(2H-l,3-Benzodioxol-5-yl)-5-

[0283] (pyridin-2-yl)-lH-imidazol-2-yl]benzamide). The TGF-Beta superfamily inhibitor may also be known as a TGF-Beta receptor kinase inhibitor.

[0284] The TGF-Beta superfamily inhibitor may be selected from a list comprising dorsomorphin dihydrochloride, LDN193189, BMPR2-IN-1 TFA , Fidrisertib , ALK2-IN-1 (activin receptor-like kinase-2

[0285] (ALK2) inhibitor described in patent publication no. WO2017181117A1, which is herein incorporated by reference), Galunisertib monohydrate, Galunisertib, RepSox, DMH-1, LY2109761, SB 525334,

[0286] LDN193189 Tetrahydrochloride, Vactosertib, Bimagrumab, A 83-01 sodium, SB-505124, LY-364947, and GW788388, or combinations thereof.

[0287] The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of about

[0288] 10 μM. The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of at least about 1 μM. In another embodiment, the TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of at least about 5 μM. The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of from about 5 μM to about 20 μM.

[0289] The c-kit receptor agonist, such as SCF (stem cell factor) may signal via c-kit receptor to activate multiple signalling pathways including MARK, ERK, JNK, NFKB, rriTOR, and JAK / STAT. The skilled person will recognise that an analogous protein could activate the c-kit receptor.

[0290] The c-kit receptor agonist, such as SCF, may be provided at a concentration of about 50 ng / ml. The c- kit receptor agonist, such as SCF, may be provided at a concentration of at least 1 picogram / mL In another embodiment, the c-kit receptor agonist, such as SCF, may be provided at a concentration of at least 30 ng / ml. The c-kit receptor agonist, such as SCF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the c-kit receptor agonist, such as SCF, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0291] Hematopoietic cell induction medium

[0292] According to another aspect of the present invention, there is provided a hematopoietic cell (HPC) induction medium, wherein the hematopoietic cell induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) an activator of VEGF signalling, such as VEGF, ii) an activator of FGF signalling, such as FGF2, iii) a c-kit receptor agonist, such as SCF, iv) an IL-3 receptor agonist, such as IL-3, v) an IL-6 receptor agonist and / or GP130 agonist, such as IL-6, and vi) a TPO (thrombopoietin) receptor agonist, such as TPO.

[0293] Each of the components i)-vi) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0294] The activator of VEGF signalling, such as VEGF, may be provided at a concentration of about 50 ng / ml.

[0295] The activator of VEGF signalling, such as VEGF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of VEGF signalling, such as VEGF, may be provided at a concentration of at least 30 ng / ml. The activator of VEGF signalling, such as VEGF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of VEGF signalling, such as VEGF, may be provided at a concentration of from about 30 ng / ml to about

[0296] 100 ng / ml.

[0297] The activator of FGF signalling, such as FGF2, may be provided at a concentration of about 50 ng / ml.

[0298] The activator of FGF signalling, such as FGF2, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of FGF signalling, such as FGF2, may be provided at a concentration of at least 30 ng / ml. The activator of FGF signalling, such as FGF2, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of FGF signalling, such as FGF2, may be provided at a concentration of from about 30 ng / ml to about

[0299] 100 ng / ml.

[0300] The c-kit receptor agonist, such as SCF, may be provided at a concentration of about 50 ng / ml. The c- kit receptor agonist, such as SCF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the c-kit receptor agonist, such as SCF, may be provided at a concentration of at least 30 ng / ml. The c-kit receptor agonist, such as SCF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the c-kit receptor agonist, such as SCF, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0301] The lL-3 receptor agonist may activate multiple signalling cascades including AKT, NFKB, MEK / ERK, STAT and SMAD signalling. The IL-3 receptor agonist may comprise synthetically produced IL-3 alternatives, such as synthokines described by Thomas et al. (Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3779-83. doi: 10.1073 / pnas.92.9.3779. PMID: 7537376; PMCID: PMC42045), which is herein incorporated by reference. The IL-3 receptor agonist may comprise SC-5S494 (comprising the sequence:

[0302] The IL-3 receptor agonist, such as IL-3, may be provided at a concentra^on of about 10 ng / ml. The IL- 3 receptor agonist, such as IL-3, may be provided at a concentra^on of at least 1 picogram / ml. In another embodiment, the IL-3 receptor agonist, such as IL-3, may be provided at a concentra^on of at least 5 ng / ml. The IL-3 receptor agonist, such as IL-3, may be provided at a concentra^on of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IL-3 receptor agonist, such as IL-3, may be provided at a concentra^on of from about 1pg / ml to 100mg / ml. In another embodiment, the IL-3 receptor agonist, such as IL-3, may be provided at a concentra^on of from about 1pg / ml to 20mg / ml. The IL-6 receptor agonist and / or GP130 agonist may signal via the IL6 receptor or GP130 to ac^vate ERK, STAT, SOCS, mediated signalling. The IL-6 receptor agonist and / or GP130 agonist may comprise a small molecule agonist, such as GP130 receptor agonist-1 having the structure: . The IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentra^on of about 50 ng / ml. The IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentra^on of at least 1 picogram / ml. In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentra^on of at least 30 ng / ml. In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentra^on of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0303] The TPO receptor agonist may bind to the TPO receptor to activate AKT, STAT, MAPK, and ERK signalling.

[0304] The TPO receptor agonist may comprise a peptide or polypeptide analogue of TPO, or a small molecule agonist. The TPO receptor agonist may comprise TPO, romiplostim or eltrombopag; or a combination thereof. In one embodiment the TPO receptor agonist comprises TPO.

[0305] The TPO receptor agonist, such as TPO, may be provided at a concentration of about 50 ng / ml. The

[0306] TPO receptor agonist, such as TPO, may be provided at a concentration of at least 1 picogram / mL In another embodiment, the TPO receptor agonist, such as TPO, may be provided at a concentration of at least 30 ng / ml. The TPO receptor agonist, such as TPO, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the TPO receptor agonist, such as TPO, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0307] T cell induction medium

[0308] The T cell induction medium to be used in step 5) may comprise: i) a basal medium, such as MEM a, ii) animal serum, such as foetal bovine serum, iii) an anti-oxidant, such as monothioglycerol, iv) a c-kit receptor agonist, such as SCF, v) an IL-7 receptor agonist, such as IL-7, vi) a TPO (thrombopoietin) receptor agonist, such as TPO, and vii) a FLT3 agonist, such as FLT3L.

[0309] Each of the components iii )-vii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0310] According to one aspect, the basal medium of the T cell induction medium may comprise MEM a. The

[0311] MEM a may be formulated in accordance with Table 6 herein. In some embodiments, the basal medium may be diluted by the other components ii)-vii) of the T cell induction medium. According to one aspect, the animal serum of the T cell induction medium may comprise or consist of foetal bovine serum. According to another aspect, the animal serum may comprise human serum.

[0312] According to one aspect, the animal serum such as foetal bovine serum may be a synthetic substitute of animal serum, expressly formulated to be an animal-free version of animal (such as foetal bovine) serum. The animal serum, such as foetal bovine serum, may be treated, e.g. heat-inactivated, and / or filtered (such as through a 0.22 pm-pore filter), and / or sterilised. The animal serum, such as foetal bovine serum, which may be treated, may be provided in the T cell induction in a volume that corresponds to from about 1% to about 20% of the volume of the basal medium of the T cell induction medium. The animal serum, such as foetal bovine serum, which may be treated, may preferably be provided in the T cell induction in a volume that corresponds to about 10% of the volume of the basal medium of the T cell induction medium.

[0313] The anti-oxidant may promote cell proliferation. The anti-oxidant may be a small molecule (e.g. less than 1 kDa). The anti-oxidant may comprise vitamin A, vitamin C, vitamin E, monothioglycerol or 2- mercaptoethanol, or any combination thereof. The anti-oxidant may comprise monothioglycerol or 2- mercaptoethanoL In one embodiment, the anti-oxidant comprises monothioglycerol.

[0314] The monothioglycerol (or thioglycerol, or 1 -thioglycerol) may comprise mono-thio glycerol. The mono- thio glycerol may initially be provided as a solution, e.g. a concentrated solution that is diluted in the

[0315] T cell induction medium. In such embodiments, the concentrated mono-thio glycerol solution may be e.g. an aqueous solution, or a solution of basal medium, such as phosphate-buffered saline (PBS) or

[0316] Dulbecco's PBS (DPBS) or MEM (such as MEM a).

[0317] Preferably, the anti-oxidant, such as mono-thio glycerol, is provided in the T cell induction medium at a concentration of about 100 μM. The anti-oxidant, such as mono-thio glycerol, may be provided in the T cell induction medium at a concentration from about 10 μM to about 1 mM. The anti-oxidant, such as mono-thio glycerol, may be provided in the T cell induction medium at a concentration of at least about 10 μM, or at least about 20 μM, or at least about 30 μM, or at least about 40 μM, or at least about 50 μM, or at least about 60 μM, or at least about 70 μM, or at least about 80 μM, or at least about 90 μM, or at least about 95 μM, or at least about 100 μM, or at least about 105 μM, or at least about 110 μM, or at least about 120 μM, or at least about 150 μM, or at least about 200 μM, or at least about 1 mM, or more. The c-kit receptor agonist, such as SCF, may be provided in the T cell induction medium at a concentration of about 10 ng / ml. The c-kit receptor agonist, such as SCF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the c-kit receptor agonist, such as

[0318] SCF, may be provided at a concentration of at least 5 ng / ml. The c-kit receptor agonist, such as SCF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the c-kit receptor agonist, such as SCF, may be provided at a concentration of from about

[0319] 5 ng / ml to about 50 ng / ml. In another embodiment, the c-kit receptor agonist, such as SCF, may be provided at a concentration of from about 5 ng / ml to about 20 ng / ml.

[0320] The IL-7 receptor agonist may activate multiple signalling cascades including Jak, STAT, and Src signalling. The IL-7 receptor agonist may comprise synthetically produced IL-7 alternatives. The IL-7 receptor agonist, such as IL-7, may be provided in the T cell induction medium at a concentration of about 25 ng / ml. The IL-7 receptor agonist, such as IL-7, may be provided at a concentration of at least

[0321] 1 picogram / ml. In another embodiment, the IL-7 receptor agonist, such as IL-7, may be provided at a concentration of at least 5 ng / ml. The IL-7 receptor agonist, such as IL-7, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IL-7 receptor agonist, such as IL-7, may be provided at a concentration of from about 15 ng / ml to about 50 ng / ml.

[0322] The TRO receptor agonist may bind to the TRO receptor to activate AKT, STAT, MARK, and ERK signalling.

[0323] The TRO receptor agonist may comprise a peptide or polypeptide analogue of TRO, or a small molecule agonist. The TRO receptor agonist may comprise TRO, romiplostim or eltrombopag; or a combination thereof. In one embodiment the TRO receptor agonist comprises TRO.

[0324] The TRO receptor agonist, such as TRO, may be provided in the T cell induction medium at a concentration of about 50 ng / ml. The TRO receptor agonist, such as TRO, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the TRO receptor agonist, such as

[0325] TRO, may be provided at a concentration of at least 30 ng / ml. The TRO receptor agonist, such as TRO, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the TRO receptor agonist, such as TRO, may be provided at a concentration of from about

[0326] 30 ng / ml to about 100 ng / ml.

[0327] The FLT3 agonist, such as FLT3L, may be provided in the T cell induction medium at a concentration of about 10 ng / ml. The FLT3 agonist, such as FLT3L, may be provided at a concentration of at least 1 picogram / mL In another embodiment, the FLT3 agonist, such as FLT3L, may be provided at a concentration of at least 5 ng / ml. The FLT3 agonist, such as FLT3L, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the FLT3 agonist, such as FLT3L, may be provided at a concentration of from about 5 ng / ml to about 50 ng / ml. In another embodiment. the FLT3 agonist, such as FLT3L, may be provided at a concentration of from about 5 ng / ml to about

[0328] 20 ng / ml.

[0329] Monocyte induction medium

[0330] According to another aspect of the present invention, there is provided a monocyte induction medium, wherein the monocyte induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) an IL-3 receptor agonist, such as IL-3, ii) an IL-6 receptor agonist and / or GP130 agonist, such as IL-6, and iii) a c-Fms receptor agonist, such as M-CSF.

[0331] Each of the components i), ii) or iii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0332] The IL-3 receptor agonist, such as IL-3, may be provided at a concentration of about 10 ng / ml. The IL-

[0333] 3 receptor agonist, such as IL-3, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the IL-3 receptor agonist, such as IL-3, may be provided at a concentration of at least 5 ng / ml. In another embodiment, the IL-3 receptor agonist, such as IL-3, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. The IL-3 receptor agonist, such as IL-3, may be provided at a concentration of lμg / ml to 20mg / ml.

[0334] The IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentration of about 50 ng / ml. The IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the IL-6 receptor agonist and / or

[0335] GP130 agonist, such as IL-6, may be provided at a concentration of at least 30 ng / ml. The IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentration of from about 1 picogram / ml to about I g / mL In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as IL-6, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml. The c-Fms receptor agonist may signal via the c-Fms receptor to activate ERK and AKT signalling. The c-Fms receptor agonist may comprise M-CSF (Macrophage Colony-Stimulating Factor). Alternatively, the c-Fms receptor agonist may comprise an antibody such as Eflapegrastim.

[0336] The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of about 80 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 1 picogram / ml.

[0337] In another embodiment, the c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 40 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of from about 40 ng / ml to about

[0338] 160 ng / ml.

[0339] Macrophage induction medium

[0340] According to another aspect of the present invention, there is provided a macrophage induction medium, wherein the macrophage induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises c-Fms receptor agonist, such as M-CSF. The M-CSF may be replaced with a peptide, protein or small molecule that will activate the same receptor or intracellular signalling pathways as M-CSF.

[0341] The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of about 80 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 1 picogram / ml.

[0342] In another embodiment, the c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 40 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of from about 40 ng / ml to about

[0343] 160 ng / ml.

[0344] Polarisation medium M0, Ml and M2

[0345] According to another aspect of the present invention, there is provided a polarisation medium M0, wherein the polarisation medium M0 comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises c-Fms receptor agonist, such as M-CSF. The M-CSF may be replaced with a peptide, protein or small molecule that will activate the same receptor or intracellular signalling pathways as M-CSF.

[0346] According to another aspect of the present invention, there is provided a polarisation medium Ml, wherein the polarisation medium Ml comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) an IFNAR agonist, such as IFN-gamma, and ii) an immunogen, such as LPS.

[0347] Each of the components i) or ii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0348] According to another aspect of the present invention, there is provided a polarisation medium M2, wherein the polarisation medium M2 comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises an IL-4 receptor agonist, such as IL-4. The IL-4 may be replaced with a peptide, protein or small molecule that will activate the same receptor or intracellular signalling pathways as IL-4.

[0349] The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of about 80 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 1 picogram / ml.

[0350] In another embodiment, the c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 40 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of from about 40 ng / ml to about

[0351] 160 ng / ml.

[0352] The IFNAR agonist may signal via IFNAR to activate stat, pi3k, AKT, and mTOR signalling. As an alternative to IFN-gamma, the skilled person will recognise that multiple alternative IFNAR agonists exist and may be used as a substitute to IFN-gamma. The IFNAR agonist may be selected from the list comprising IFN-gamma, KRN7000 analog 1, RO8191 (CDM-3008), interferon alfa, interferon receptor inducer-1, and SM-276001; or combinations thereof.

[0353] The KRN7000 analog 1 may have the structure:

[0354]

[0355] The RO8191 (CDM-3008) may have the structure:

[0356] The interferon receptor inducer- 1 may have the structure:

[0357] SM-276001 may have the structure:

[0358] The IFNAR agonist, such as IFN-gamma, may be provided at a concentration of about 20 ng / ml. The

[0359] IFNAR agonist, such as IFN-gamma, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the IFNAR agonist, such as IFN-gamma, may be provided at a concentration of at least 10 ng / ml. The IFNAR agonist, such as IFN-gamma, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IFNAR agonist, such as IFN-gamma, may be provided at a concentration of from about 10 ng / ml to about 40 ng / ml.

[0360] The immunogen may stimulate an immune response via toll-like receptors, such as TLR4 for example for LPS. The signalling may be via N FKB to cause inflammation. In one embodiment, the immunogen comprises LPS. The skilled person will recognise that any non-human stimuli could be used as a substitute for LPS. For example, viral proteins, viral RNA or DNA, human cellular debris from dead cells, or extracellular matrix components, such as partially degraded collagen. These immunogens can be classified as pathogen associated molecular patterns (PAMPs) of Damage Associated Molecular

[0361] Patterns (DAMPs).

[0362] The immunogen, such as LPS, may be provided at a concentration of about 100 ng / ml. The immunogen, such as LPS, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the immunogen, such as LPS, may be provided at a concentration of at least 50 ng / ml.

[0363] The immunogen, such as LPS, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the immunogen, such as LPS, may be provided at a concentration of from about 50 ng / ml to about 200 ng / ml.

[0364] The IL-4 receptor agonist, such as IL-4, may be provided at a concentration of about 20 ng / ml. The IL-

[0365] 4 receptor agonist, such as IL-4, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the IL-4 receptor agonist, such as IL-4, may be provided at a concentration of at least 10 ng / ml. The IL-4 receptor agonist, such as IL-4, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IL-4 receptor agonist, such as IL-4, may be provided at a concentration of from about 10 ng / ml to about 40 ng / ml.

[0366] Immature dendritic cell induction medium

[0367] According to another aspect of the present invention, there is provided an immature dendritic cell induction medium, wherein the immature dendritic cell induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a GM-CSF receptor agonist, such as GM-CSF, and ii) an IL-4 receptor agonist, such as IL-4. Each of the components i) or ii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0368] The GM-CSF receptor agonist may signal via the GM-receptorto activate Jak / STAT / AKT / GSK3 signalling.

[0369] The GM-CSF receptor agonist may comprise GM-CSF, or a mimetic thereof, such as a peptide mimetic.

[0370] US patent publication no. US11267859B2 (which is incorporated herein by reference) identifies suitable peptide mimetics of GM-CSF, which may be used as an alternative to GM-CSF.

[0371] The GM-CSF receptor agonist, such as GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), may be provided at a concentration of about 50 ng / ml. The GM-CSF receptor agonist, such as GM-CSF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the GM-CSF may be provided at a concentration of at least 30 ng / ml. The GM-CSF receptor agonist, such as GM-

[0372] CSF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the GM-CSF receptor agonist, such as GM-CSF, may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0373] The IL-4 receptor agonist may activate STAT signalling pathways. The IL-4 receptor agonist, such as IL-

[0374] 4, may be provided at a concentration of about 20 ng / ml. The IL-4 receptor agonist, such as IL-4, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the IL-4 receptor agonist, such as IL-4, may be provided at a concentration of at least 10 ng / ml. The IL-4 receptor agonist, such as IL-4, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IL-4 receptor agonist, such as IL-4, may be provided at a concentration of from about 10 ng / ml to about 40 ng / mL

[0375] Mature dendritic cell induction medium

[0376] According to another aspect of the present invention, there is provided a mature dendritic cell induction medium, wherein the mature dendritic cell induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a TNF receptor activator, such as TNF-alpha, and ii) an immunogen, such as LPS. Each of the components i) or ii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0377] The TNF receptor activator may activate NFKB signalling. In one embodiment, the TNF receptor activator comprises TNF-alpha. Alternative receptor and pathway activators may comprise those selected from Oxazolone, Bioymifi, GSK2245035, Lornoxicam (Chlortenoxicam), Beta-Eudesmol, LY

[0378] 303511 hydrochloride, HDMAPP triammonium, Cetrelimab, (E)-C-HDMAPP ammonium, LY 303511 dihydrochloride, D-Trimannuronic acid, and LY 303511; or combinations thereof.

[0379] The TNF receptor activator, such as TNF-alpha, may be provided at a concentration of about 100 ng / ml.

[0380] The TNF receptor activator, such as TNF-alpha, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the TNF receptor activator, such as TNF-alpha, may be provided at a concentration of at least 50 ng / ml. The TNF receptor activator, such as TNF-alpha, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the TNF receptor activator, such as TNF-alpha, may be provided at a concentration of from about 50 ng / ml to about 200 ng / ml.

[0381] The immunogen, such as LPS, may be provided at a concentration of about 100 ng / ml. The immunogen, such as LPS, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the immunogen, such as LPS, may be provided at a concentration of at least 50 ng / ml.

[0382] The immunogen, such as LPS, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the immunogen, such as LPS, may be provided at a concentration of from about 50 ng / ml to about 200 ng / ml.

[0383] Endoderm induction medium

[0384] According to another aspect of the present invention, there is provided an endoderm induction medium, wherein the endoderm induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a Wnt signalling activator, such as CHIR99021, ii) a PI3K inhibitor, such as LY294002, and iii) an activator of SMAD2 / 3 signalling, such as Activin A. Each of the components i), ii) or iii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0385] The Wnt signalling activator may be a small molecule inhibitor (i..e. <900Da) of the inhibitor of Wnt signalling (GSK3b), such as CHIR99021. In another embodiment, the Wnt signalling activator may be a peptide activator, such as Wnt3a, or a functionally equivalent peptide thereof having the function of a

[0386] 0-catenin pathway agonist. In one embodiment, the Wnt signalling activator is CHIR99021. CHIR99021 may be provided at a concentration of about 3 μM.

[0387] The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of at least 1 μM.

[0388] In another embodiment, the Wnt signalling activator, such as CHIR99021, may be provided at a concentration of at least 2 μM. The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the Wnt signalling activator. such as CHIR99021, may be provided at a concentration of from about 2 μM to about 6 μM. The Wnt signalling activator, such as CHIR99021, may only be provided in day 1 (DI) of the culture, for example if the endoderm induction medium is changed / refreshed after 12-24 hours, the Wnt signalling activator, such as CHIR99021, may not be provided in the new endoderm induction medium.

[0389] The PI3K inhibitor may be as described herein. The PI3K inhibitor, such as LY294002, may be provided at a concentration of about 10 μM. The PI3K inhibitor, such as LY294002, may be provided at a concentration of at least about 1 μM. In another embodiment, the PI3K inhibitor, such as LY294002, may be provided at a concentration of at least about 5 μM. The PI3K inhibitor, such as LY294002, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the PI3K inhibitor, such as LY294002, may be provided at a concentration of from about 5 μM to about 15 μM.

[0390] The PI3K inhibitor, such as LY294002, may only be provided in day 1 (DI) and day 2 (D2) of the culture, for example if the endoderm induction medium is changed / refreshed every 12-24 hours, the PI3K inhibitor, such as LY294002, may not be provided in the third endoderm induction medium.

[0391] The activator of SMAD2 / 3 signalling may be as described herein. The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of about 100 ng / ml. The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of at least 50 ng / ml. The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of from about 50 ng / ml to about 200 ng / ml.

[0392] Anterior foregut endoderm induction medium

[0393] According to another aspect of the present invention, there is provided an anterior foregut endoderm induction medium, wherein the anterior foregut endoderm induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a BMP pathway inhibitor, such as dorsomorphin, and ii) a TGF-Beta superfamily inhibitor, such as SB431542.

[0394] Each of the components i) or ii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0395] The BMP pathway inhibitor may comprise a small molecule inhibitor, such as selected from A 77-01

[0396] (4-[3-(6-Methyl-2-pyridinyl)-lH-pyrazol-4-yl]quinoline), A 83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-

[0397] (4-quinolinyl)-lH-pyrazole-l-carbothioamide), DMH-1 (4-[6-[4-(l-Methylethoxy)phenyl]pyrazolo[l,5- a]pyrimidin-3-yl]-quinoline), DMH2 (4-[6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]pyrazolo[l,5- a]pyrimidin-3-yl]quinoline), Dorsomorphin dihydrochloride (6-[4-[2-(l-Piperidinyl)ethoxy]phenyl]-3-

[0398] (4-pyridinyl)-pyrazolo[l,5-a]pyrimidine dihydrochloride), K 02288 (3-[(6-Amino-5-(3,4,5- trimethoxyphenyl)-3-pyridinyl]phenol), LDN 193189 dihydrochloride (4-[6-[4-(l-

[0399] Piperazinyl)phenyl]pyrazolo[l,5-a]pyrimidin-3-yl]quinoline dihydrochloride), M4K2163 dihydrochloride (l-[4-[5-(4-Fluoro-3,5-dimethoxyphenyl)-4-methyl-3-pyridinyl]phenyl]piperazine dihydrochloride), ML 347 (5-[6-(4-Methoxyphenyl)pyrazolo[l,5-a]pyrimidin-3-yl]quinoline), and SB

[0400] 505124 (2-[4-(l,3-Benzodioxol-5-yl)-2-(l,l-dimethylethyl)-lH-imidazol-5-yl]-6-methyl-pyridine), or combinations thereof.

[0401] The BMP pathway inhibitor may comprise a protein such as noggin.

[0402] The BMP pathway inhibitor, such as Dorsomorphin, may be provided at a concentration of about 2 pm.

[0403] The BMP pathway inhibitor, such as Dorsomorphin, may be provided at a concentration of at least 1 μM. In another embodiment, the BMP pathway inhibitor, such as Dorsomorphin, may be provided at a concentration of at least 1 μM. The BMP pathway inhibitor, such as Dorsomorphin, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the BMP pathway inhibitor, such as Dorsomorphin, may be provided at a concentration of from about 1 μM to about 15 μM.

[0404] The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of about

[0405] 10 pm. The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of at least 1 μM. In another embodiment, the TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of at least 5 μM. The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the

[0406] TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of from about

[0407] 5 μM to about 15 μM.

[0408] Lung progenitor cell induction medium

[0409] According to another aspect of the present invention, there is provided a lung progenitor cell induction medium, wherein the lung progenitor cell induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a Wnt signalling activator, such as Wnt3A, ii) an agent to potentiate Wnt signalling, such as R-Spondinl, iii) a BMP pathway activator, such as BMP4, and iv) a retinoic acid receptor agonist, such as retinoic acid.

[0410] Each of the components i), ii), iii) or iv) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0411] The Wnt signalling activator may comprise Wnt3a, or a functionally equivalent peptide thereof having the function of a 0-catenin pathway agonist. In one embodiment, the Wnt signalling activator is

[0412] CHIR99021.

[0413] The Wnt signalling activator, such as Wnt3A, may be provided at a concentration of about 100 ng / ml.

[0414] The Wnt signalling activator, such as Wnt3A, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the Wnt signalling activator, such as Wnt3A, may be provided at a concentration of at least 50 ng / ml. The Wnt signalling activator, such as Wnt3A, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the Wnt signalling activator, such as Wnt3A, may be provided at a concentration of from about 50 ng / ml to about 200 ng / ml.

[0415] The agent to potentiate Wnt signalling may comprise R-Spondinl. R-Spondinl (RSPO1) is the prototypic member of the R-spondin family and is used to potentiate Wnt signaling in many organoid culture systems including intestinal and tumor (cancer) organoid culture. R-spondin 1 is also required for hematopoietic stem cell specification and cancer cell migration and survival. Alternative to RSPO1 are other r-spondin family members, such as R-spondin 1,2,3, or 4.

[0416] The agent to potentiate Wnt signalling, such as R-Spondinl, may be provided at a concentration of about 250 ng / ml. The agent to potentiate Wnt signalling, such as R-Spondinl, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the agent to potentiate Wnt signalling, such as R-Spondinl, may be provided at a concentration of at least 200 ng / ml. The agent to potentiate Wnt signalling, such as R-Spondinl, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the agent to potentiate Wnt signalling, such as R-Spondinl, may be provided at a concentration of from about 200 ng / ml to about 300 ng / ml.

[0417] The BMP pathway activator may be as described herein above. The BMP pathway activator, such as

[0418] BMP4, may be provided at a concentration of about 10 ng / ml. The BMP pathway activator, such as

[0419] BMP4, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the

[0420] BMP pathway activator, such as BMP4, may be provided at a concentration of at least 5 ng / ml. The

[0421] BMP pathway activator, such as BMP4, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of from about 5 ng / ml to about 20 ng / ml.

[0422] The retinoic acid receptor agonist may comprise retinoic acid or analogues thereof. Retinoic acid (RA) binds to RA receptor (RAR) in an RAR-retinoid X receptor (RXR) heterodimer complex bound to RA response elements (RAREs) near target genes, resulting in control of transcription. Alternative retinoic acid receptor agonists may be selected from AC 261066 (4-[4-(2-Butoxyethoxy-)-5-methyl-2-thiazolyl]-

[0423] 2-fluorobenzoic acid), Adapalene (6-(4-Methoxy-3-tricyclo[3.3.1.13,7]dec-l-ylphenyl)-2- naphthalenecarboxylic acid). AM 580 (4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carboxamido]benzoic acid), AM 80 (4-[[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)amino]carbonyl]benzoic acid), BMS 753 (4-[[(2,3-Dihydro-l,l,3,3-tetramethyl-2-oxo-lH- inden-5-yl)carbonyl]amino]benzoic acid), BMS 961 (3-Fluoro-4-[[2-hydroxy-2-(5,5,8,8-tetramethyl-

[0424] 5,6,7,8,-tetrahydro-2-naphthalenyl)acetyl]amino]-benzoic acid), CD 1530 (4-(6-Hydroxy-7 tricyclo[3.3.1.13,7]dec-l-yl-2-naphthalenyl)benzoic acid), CD 2314 (5-(5,6,7,8-Tetrahydro-5,5,8,8- tetramethyl-2-anthracenyl)-3-thiophenecarboxylic acid), CD 437 (6-(4-Hydroxy-3- tricyclo[3.3.1.13,7]dec-l-ylphenyl)-2-naphthalenecarboxylic acid), Ch 55 (4-[(lE)-3-[3,5-bis(l,l-

[0425] Dimethylethyl)phenyl]-3-oxo-l-propenyl]benzoic acid), DC 271 (4-[2-[l,2,3,4-Tetrahydro-4,4-dimethyl- l-(l-methylethyl)-6-quinolinyl]ethynyl]benzoic acid), and TTNPB (4-[(E)-2-(5, 6,7, 8-Tetrahydro-5, 5,8,8- tetramethyl-2-naphthalenyl)-l-propenyl]benzoic acid), or combinations thereof.

[0426] The retinoic acid receptor agonist, such as retinoic acid, may be provided at a concentration of about

[0427] 100 nM. The retinoic acid receptor agonist, such as retinoic acid, may be provided at a concentration of at least 1 μM. In another embodiment, the retinoic acid receptor agonist, such as retinoic acid, may be provided at a concentration of at least 50 nM. The retinoic acid receptor agonist, such as retinoic acid, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the retinoic acid receptor agonist, such as retinoic acid, may be provided at a concentration of from about 50 nM to about 200 nM.

[0428] Distal lung maturation induction medium

[0429] According to another aspect of the present invention, there is provided a distal lung maturation induction medium, wherein the distal lung maturation induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) Wnt signalling activator, such as CHIR99021, ii) a FGF receptor agonist, such as FGF10, iii) a FGFR2II IB receptor agonist, such as FGF7, iv) a phosphodiesterase inhibitor, such as IBMX (3-isobutyl-l-methylxanthine), v) an activator of PKA signalling, such as cAMP or 8Bro-cAMP, and vi) a corticosteroid, such as DEXA or hydrocortisone.

[0430] Each of the components i), ii), iii), iv), v) orvi) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways. The Wnt signalling activator may be a small molecule inhibitor (i..e. <900Da) of the inhibitor of Wnt signalling (GSK3b), such as CHIR99021. In another embodiment, the Wnt signalling activator may be a peptide activator, such as Wnt3a, or a functionally equivalent peptide thereof having the function of a

[0431] 0-catenin pathway agonist. In one embodiment, the Wnt signalling activator is CHIR99021. The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of about 3 pm. The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of at least 1 μM. In another embodiment, the Wnt signalling activator, such as CHIR99021, may be provided at a concentration of at least 2 μM. The Wnt signalling activator, such as CHIR99021, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the Wnt signalling activator, such as

[0432] CHIR99021, may be provided at a concentration of from about 2 μM to about 6 μM.

[0433] The FGF receptor agonist, such as FGF1O (also known as KGF) may work via the FGF receptors including

[0434] FGFR1 and FGFR2 to activate MARK signalling, AKT, ERKs and JNK signal pathways. The skilled person will recognise that other FGFs or KGFs could be used in place of FGF1O for example FGF2. Other FGFR agonists include Efruxifermin and Fazpilodemab.

[0435] The FGF receptor agonist, such as FGF10, may be provided at a concentration of about 10 ng / ml. The

[0436] FGF receptor agonist, such as FGF1O, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the FGF receptor agonist, such as FGF10, may be provided at a concentration of at least 5 ng / ml. The FGF receptor agonist, such as FGF10, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the FGF receptor agonist, such as

[0437] FGF10, may be provided at a concentration of from about 5 ng / ml to about 20 ng / ml.

[0438] The FGFR2IIIB receptor agonist may comprise FGF7. The FGFR2IIIB receptor agonist may activate

[0439] MARK, ERK, AKT, and JNK signalling. Other FGF family member could replace FGF7 to achieve similar effects. FGF7 may be substituted by FGFR2IIIB receptor agonist peptides such as hexafins described by

[0440] Li et al. (Dev Neurobiol. 2009 Nov;69(13):837-54. doi: 10.1002 / dneu.20740. PMID: 19634127), which is herein incorporated by reference.

[0441] The FGFR2IIIB receptor agonist, such as FGF7, may be provided at a concentration of about 10 ng / ml.

[0442] The FGFR2IIIB receptor agonist, such as FGF7, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the FGFR2IIIB receptor agonist, such as FGF7, may be provided at a concentration of at least 5 ng / ml. The FGFR2II I B receptor agonist, such as FGF7, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the FGFR2IIIB receptor agonist, such as FGF7, may be provided at a concentration of from about 5 ng / ml to about 20 ng / ml.

[0443] The phosphodiesterase inhibitor may be both a competitive non-selective phosphodiesterase inhibitor, which raises intracellular cAMP, activates PKA, inhibits TNFa and leukotriene synthesis. The phosphodiesterase inhibitor may further reduce inflammation and innate immunity and be a nonselective adenosine receptor antagonist. The phosphodiesterase inhibitor may comprise IBMX. In one embodiment, the phosphodiesterase inhibitor may comprise a xanthine derivative, such as caffeine.

[0444] The phosphodiesterase inhibitor, such as IBMX, may be provided at a concentration of about 0.1 mM.

[0445] The phosphodiesterase inhibitor, such as IBMX, may be provided at a concentration of at least 1 μM.

[0446] In another embodiment, the phosphodiesterase inhibitor, such as IBMX, may be provided at a concentration of at least 0.05 mM. The phosphodiesterase inhibitor, such as IBMX, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the phosphodiesterase inhibitor, such as IBMX, may be provided at a concentration of from about 0.05 mM to about 0.2 mM.

[0447] The activator of PKA signalling, such as cAMP or SBro-cAMP, may be provided at a concentration of about 0.1 mM. The activator of PKA signalling, such as cAMP or SBro-cAMP, may be provided at a concentration of at least 1 μM. In another embodiment, the activator of PKA signalling, such as cAMP or SBro-cAMP, may be provided at a concentration of at least 0.05 mM. The activator of PKA signalling, such as cAMP or SBro-cAMP, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the activator of PKA signalling, such as cAMP or SBro-cAMP, may be provided at a concentration of from about 0.05 mM to about 0.2 mM.

[0448] The corticosteroid, such as DEXA (dexamethasone) or hydrocortisone, may be provided at a concentration of about 50 nM. The corticosteroid, such as DEXA or hydrocortisone, may be provided at a concentration of at least 1 μM. In another embodiment, the corticosteroid, such as DEXA or hydrocortisone, may be provided at a concentration of at least 30 nM. The corticosteroid, such as DEXA or hydrocortisone, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the corticosteroid, such as DEXA or hydrocortisone, may be provided at a concentration of from about 30 nM to about 100 nM.

[0449] Foregut induction medium According to another aspect of the present invention, there is provided a foregut induction medium, wherein the foregut induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises an activator of SMAD2 / 3 signalling, such as Activin A.

[0450] The activator of SMAD2 / 3 signalling, such as Activin A, may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0451] The activator of SMAD2 / 3 signalling, such as Activin A, or a molecule having equivalent activity. As for

[0452] BMP, Activin A is part of the transforming growth factor beta (TGF-P) superfamily. Any of the activins, or TGF-[3s may substitute for Activin A. Similarly, the GDF subfamily can also substitute Activin A. The intracellular signalling pathway of interest is SMAD2 / 3 so any other protein and / or small molecule activating SMAD2 / 3 signalling could substitute Activin A. An example small molecule that activates

[0453] SMAD2 / 3 signalling includes SRI-011381-hydrochloride:

[0454] H-Cl

[0455] The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of about

[0456] 50 ng / ml. The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as activin

[0457] A, may be provided at a concentration of at least 30 ng / ml. The activator of SMAD2 / 3 signalling, such as activin A, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as activin A may be provided at a concentration of from about 30 ng / ml to about 100 ng / ml.

[0458] Hepatic endoderm induction medium

[0459] According to another aspect of the present invention, there is provided a hepatic endoderm induction medium, wherein the hepatic endoderm induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a TGF-Beta superfamily inhibitor, such as SB431542, ii) a BMP pathway activator, such as BMP4, iii) a FGF receptor agonist, such as FGF10.

[0460] Each of the components i), ii), or iii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0461] The TGF-Beta superfamily inhibitor may inhibit signalling via SMAD2 / 3. In one embodiment, the TGF-

[0462] Beta superfamily inhibitor comprises SB431542 (also known as 4-[4-(2H-l,3-Benzodioxol-5-yl)-5-

[0463] (pyridin-2-yl)-lH-imidazol-2-yl]benzamide). The TGF-Beta superfamily inhibitor may also be known as a TGF-Beta receptor kinase inhibitor.

[0464] The TGF-Beta superfamily inhibitor may be selected from a list comprising dorsomorphin dihydrochloride, LDN193189, BMPR2-IN-1 TFA , Fidrisertib , ALK2-IN-1 (activin receptor-like kinase-2

[0465] (ALK2) inhibitor described in patent publication no. WO2017181117A1, which is herein incorporated by reference), Galunisertib monohydrate, Galunisertib, RepSox, DMH-1, LY2109761, SB 525334,

[0466] LDN193189 Tetrahydrochloride, Vactosertib, Bimagrumab, A 83-01 sodium, SB-505124, LY-364947, and GW788388, or combinations thereof.

[0467] The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of about

[0468] 10 μM. The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of at least about 1 μM. In another embodiment, the TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of at least about 5 μM. The TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of from about 1 μM to about 1 M. In another embodiment, the TGF-Beta superfamily inhibitor, such as SB431542, may be provided at a concentration of from about 5 μM to about 20 μM.

[0469] The BMP pathway activator may activate the BMP pathway via the BMP receptors and may activate

[0470] SMAD and MARK mediated intracellular signalling cascades to affect gene expression. The skilled person will recognise that BMP4 is one such BMP pathway activator, yet multiple other BMPs can activate similar signalling in cells that may either entirely or partially overlap with the effect on BMP4.

[0471] This may include any of the BMP family of proteins, e.g. BMP1-15. Any other BMP recombinant protein, either full length or partial that is able to bind the receptor could be used instead of BMP4.

[0472] Also, small molecule BMP pathway activators are available, which are both naturally occurring and synthesised may activate the BMP pathway, for example SB 4 (2-[[(4-

[0473] Bromophenyl)methyl]thio]benzoxazole) is known to activate canonical BMP signalling and increases

[0474] SMAD-1 / 5 / 9 phosphorylation.

[0475] The BMP pathway activator, such as BMP4, may be provided at a concentration of about 50 ng / ml. The

[0476] BMP pathway activator, such as BMP4, may be provided at a concentration of at least 1 picogram / ml.

[0477] In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of at least 30 ng / ml. The BMP pathway activator, such as BMP4, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of from about 30 ng / ml to about

[0478] 100 ng / ml.

[0479] The FGF receptor agonist, such as FGF10 (also known as KGF) may work via the FGF receptors including

[0480] FGFR1 and FGFR2 to activate MAPK signalling, AKT, ERKs and JNK signal pathways. The skilled person will recognise that other FGFs or KGFs could be used in place of FGF10 for example FGF2. Other FGFR agonists include Efruxifermin and Fazpilodemab.

[0481] The FGF receptor agonist, such as FGF10, may be provided at a concentration of about 20 ng / ml. The

[0482] FGF receptor agonist, such as FGF10, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the FGF receptor agonist, such as FGF10, may be provided at a concentration of at least 2 ng / ml. The FGF receptor agonist, such as FGF10, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the FGF receptor agonist, such as

[0483] FGF10, may be provided at a concentration of from about 2 ng / ml to about 40 ng / ml.

[0484] The hepatic maturation medium

[0485] According to another aspect of the present invention, there is provided a hepatic maturation medium. wherein the hepatic maturation medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) an IL-6 receptor agonist and / or GP130 agonist, such as oncostatin M (One M, or

[0486] OSM), ii) a hepatocyte growth factor (HGF) receptor agonist, such as HGF. Each of the components i) or ii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0487] The IL-6 receptor agonist and / or GP130 agonist, such as oncostatin M (One M, or OSM) may signal to activate the Jak / STAT and Tyk2 pathways. The IL-6 receptor agonist and / or GP130 agonist, such as One

[0488] M, may be provided at a concentration of about 30 ng / ml. The IL IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of at least 15 ng / ml. The IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of from about 10 ng / ml to about 80 ng / ml.

[0489] The hepatocyte growth factor (HGF) receptor agonist, such as HGF, may signal by GABI to activate

[0490] PI3K, SHP2, and PLC-y signalling, and may further activate other growth- and / or proliferation- promoting pathways. The HGF receptor agonist, such as HGF, may be provided at a concentration of about 50 ng / ml. The HGF receptor agonist, such as HGF, may be provided at a concentration of at least

[0491] 1 picogram / ml. In another embodiment, the HGF receptor agonist, such as HGF, may be provided at a concentration of at least 30 ng / ml. The HGF receptor agonist, such as HGF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the HGF receptor agonist, such as HGF, may be provided at a concentration of from about 20 ng / ml to about

[0492] 100 ng / ml.

[0493] Cardiomyocyte induction medium

[0494] Cardiomyocytes (CMCs) may be derived from hiPSCs by culturing hiPSCs in a series of media obtained according to the invention herein. Therefore, according to another aspect of the present invention. there is provided a CMC induction medium, which may be used for cell culture to obtain CMCs from hiPSCs.

[0495] The hiPSCs whence the CMCs are to be derived may be cultured in hiPSC maintenance medium, such as E8, prior to commencement of CMC induction. The hiPSC maintenance medium may comprise any medium that is suitable to the indefinite, or long-term, or medium-term, or short-term, culturing of pluripotent stem cells, such as E8 as defined hereinabove. The hiPSCs may be cultured in hiPSC maintenance medium for an appropriate number of days before commencement of CMC induction, such as a number of days sufficient for the hiPSCs to achieve a desired cell density e.g. 70%, 80%, or more. The hiPSCs may be cultured in hiPSC maintenance medium for about 1-4 days. Preferably, the hiPSCs may be cultured in hiPSC maintenance medium for about 1-3 days. More preferably, the hiPSCs may be cultured in hiPSC maintenance medium for about 1-2 days. Most preferably, the hiPSCs may be cultured in hiPSC maintenance medium for about 2 days.

[0496] According to one aspect of the present invention, there is provided a CMC induction medium, wherein the CMC induction medium comprises the chemically defined universal medium according to the invention, and wherein the chemically defined universal medium further comprises: i) a BMP pathway activator, such as BMP4, ii) an activator of SMAD2 / 3 signalling, such as Activin A, and iii) one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, and iv) optionally an extracellular matrix (ECM) growth factor cocktail, such as Matrigel™.

[0497] Each of the components i), ii), or iii) may be replaced with a peptide, protein or small molecule that would activate the same receptor or intracellular signalling pathways.

[0498] The BMP pathway activator may activate the BMP pathway via the BMP receptors and may activate

[0499] SMAD and MAPK mediated intracellular signalling cascades to affect gene expression. The skilled person will recognise that BMP4 is one such BMP pathway activator, yet multiple other BMPs can activate similar signalling in cells that may either entirely or partially overlap with the effect on BMP4.

[0500] This may include any of the BMP family of proteins, e.g. BMP1-15. Any other BMP recombinant protein, either full length or partial that is able to bind the receptor could be used instead of BMP4.

[0501] Also, small molecule BMP pathway activators are available, which are both naturally occurring and synthesised may activate the BMP pathway, for example SB 4 (2-[[(4-

[0502] Bromophenyl)methyl]thio]benzoxazole) is known to activate canonical BMP signalling and increases

[0503] SMAD-1 / 5 / 9 phosphorylation.

[0504] The BMP pathway activator, such as BMP4, may be provided at a concentration of about 1 ng / ml. The

[0505] BMP pathway activator, such as BMP4, may be provided at a concentration of at least 1 picogram / ml.

[0506] In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of at least 800 μg / ml. The BMP pathway activator, such as BMP4, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of from about 800 μg / ml to about 1.5 ng / ml.

[0507] In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of about 10 ng / ml. The BMP pathway activator, such as BMP4, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the BMP pathway activator, such as

[0508] BMP4, may be provided at a concentration of at least 5 ng / ml. The BMP pathway activator, such as

[0509] BMP4, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the BMP pathway activator, such as BMP4, may be provided at a concentration of from about 8 ng / ml to about 25 ng / ml.

[0510] The BMP pathway activator, such as BMP4, may only be provided for the first three days of culture in the CMC induction medium. In particular embodiments, the BMP pathway activator, such as BMP4, may be provided in the CMC induction medium at different concentrations on different days of culture.

[0511] For example, if the BMP pathway activator, such as BMP4, is provided at a lower concentration (such as about 1 ng / ml) on the first day of culture in the CMC induction medium (day = D2), and the CMC medium is refreshed the day after (D3), then on refreshing the CMC induction medium the BMP pathway activator, such as BMP4, may be provided in the new CMC induction medium at a higher concentration (such as about 10 ng / ml).

[0512] The ECM growth factor cocktail, such as Matrigel™, may be for coating the culture flask. It may comprise dissolving the ECM growth factor cocktail, such as Matrigel™, in media to be added to the culture flask. The ECM growth factor cocktail, such as Matrigel™, may be dissolved, redissolved, or diluted in the CDUM according to the invention. Preferably, the ECM growth factor cocktail, such as

[0513] Matrigel™, is diluted in the CDUM at a ratio of about 0.25-10% (v:v) (ECM growth factor cocktail :

[0514] CDUM). More preferably, the ECM growth factor cocktail, such as Matrigel™, is diluted in the CDUM at a ratio of about 0.5-5% (v:v) (ECM growth factor cocktail : CDUM). Yet more preferably, the ECM growth factor cocktail, such as Matrigel™, is diluted in the CDUM at a ratio of about 0.5-2% (v:v) (ECM growth factor cocktail : CDUM). Most preferably, the ECM growth factor cocktail, such as Matrigel™, is diluted in the CDUM at a ratio of about 1% (v:v) (ECM growth factor cocktail : CDUM). The person skilled in the art will notice that the concentration of components in the CDUM will be slightly diluted by the addition of the ECM growth factor cocktail, such as Matrigel™, and will therefore understand that the

[0515] ECM growth factor cocktail, such as Matrigel™, may be provided in a dilution that balances the provision of ECM growth factors and yet maintains acceptable concentrations of CDUM components. The ECM growth factor cocktail, such as Matrigel™, may only be provided for the first day of culture in the CMC induction medium. For example, if the ECM growth factor cocktail, such as Matrigel™, is provided on the first day of culture in the CMC induction medium (day = D2), and the CMC medium is refreshed the day after (D3), then on refreshing the CMC induction medium the ECM growth factor cocktail, such as Matrigel™, may be not provided in the new CMC induction medium.

[0516] The skilled person will be familiar with ECMs and alternatives to Matrigel™, which may be used, such as collagen, laminin, a hydrogel, a polymer. In one embodiment, the ECM comprises or consists of collagen, laminin, a hydrogel, or polymer; or combinations thereof. In one embodiment, the ECM comprises or consists of laminin.

[0517] The activator of SMAD2 / 3 signalling, such as Activin A, or a molecule having equivalent activity. As for

[0518] BMP, Activin A is part of the transforming growth factor beta (TGF-[3) superfamily. Any of the activins, or TGF-0S may substitute for Activin A. Similarly, the GDF subfamily can also substitute Activin A. The intracellular signalling pathway of interest is SMAD2 / 3 so any other protein and / or small molecule activating SMAD2 / 3 signalling could substitute Activin A. An example small molecule that activates

[0519] SMAD2 / 3 signalling includes SRI-011381-hydrochloride:

[0520] A 1 J

[0521] N N '' i H

[0522] H-CI

[0523] The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of aboutS ng / ml. The activator of SMAD2 / 3 signalling, such as Activin A, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as activin

[0524] A, may be provided at a concentration of at least 3 ng / ml. The activator of SMAD2 / 3 signalling, such as activin A, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the activator of SMAD2 / 3 signalling, such as activin A may be provided at a concentration of from about 3 ng / ml to about 20 ng / ml. The activator of SMAD2 / 3 signalling, such as

[0525] Activin A, may only be provided on second day of culture in the CMC induction medium (day = D3). For example, if the activator of SMAD2 / 3 signalling, such as Activin A, is provided on the second day of culture in the CMC induction medium (day = D3), and the CMC medium is refreshed two days after (D5), then on refreshing the CMC induction medium the activator of SMAD2 / 3 signalling, such as

[0526] Activin A, may be not provided in the new CMC induction medium.

[0527] The one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, may be provided at a concentration of about 10 μM each. The one or more inhibitors of Wnt signalling, such as KY0211 and

[0528] XAV939, may be provided at a concentration of at least 1 μM each. In another embodiment, the one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, may be provided at a concentration of at least 3 μM each. The one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, may be provided at a concentration of from about 1 μM each to about 1 M each. In another embodiment, the one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, may be provided at a concentration of from about 3 μM each to about 20 μM each. In a preferred embodiment, both KY0211 and AXV939 are provided in the CMC induction medium.

[0529] The CMC induction medium may comprise one, or more, inhibitors of Wnt signalling, such as KY0211 and XAV939. It may be advantageous for the CMC induction medium to comprise more than one inhibitor of Wnt signalling, such as KY0211 and XAV939 e.g. because one such inhibitor is unlikely to effectively inhibit all strands of the Wnt signalling pathway. KY0211 may have the following chemical structure:

[0530] XAV939 may have the following chemical structure:

[0531] By way of example, while XAV939 is understood to be a potent inhibitor of TNKS1 and TNKS2, it is unlikely that XAV939 also behaves as an efficacious inhibitor of canonical Wnt signalling and to induce downregulation of Wnt signalling target genes, which are understood to be the effects of KY0211 treatment, and vice versa. Therefore, in one embodiment, the CMC induction medium comprises one or more inhibitors of Wnt signalling, whereby the said inhibitors collectively inhibit TNKS1 / 2 and canonical Wnt signalling targets. Although KY0211 and XAV939 are here singled out, it is expressly envisaged that structural and / or functional homologues thereof may also, or isntead, be comprised by the CMC induction medium. Therefore, in one embodiment, the CMC induction medium comprises structural and / or functional homologues of KY0211 and / or XAV939.

[0532] The one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, may only be provided to the cells after three days of culture in the CIVIC induction medium (day = D5). The one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, may then be refreshed after about 2 days to ensure they are present in the CMC induction medium at an active concentration. For example, if one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, are provided after about 3 days of culture in the CMC induction medium (day = D5), and the CMC medium is refreshed two days after (D7), then on refreshing the CIVIC induction medium the one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, may be provided again in the new CMC induction medium.

[0533] Cardiomyocyte maturation medium

[0534] To obtain mature CMCs, culture in the CMC induction medium may be followed by culture of the cells in CMC maturation medium. The CMC maturation medium may be any medium that allows the growth and proliferation of relatively immature CMC and their phenotypic switch to mature CMCs. By way of example, the CMC maturation medium may comprise CDUM.

[0535] The assembloid aggregation medium

[0536] Assembloids, such as multi-tissue liver assembloids (mLAs), may be assembled by culturing cells in an assembloid aggregation medium for period of time defined as the assembloid aggregation period. In the assembloid aggregation period, cells may be cultured in an appropriate culture medium that allows cell growth, such as CDUM, or more preferably CDEM. In some embodiments the culture medium of the assembloid aggregation period may comprise CDUM, or more preferably CDEM and supplements.

[0537] In some embodiments, such as embodiments comprising mLAs, the supplements may comprise: a hepatocyte growth factor (HGF) receptor agonist, such as HGF, ii. an IL-6 receptor agonist and / or GP130 agonist, such as oncostatin M (One M, or OSM), iii. a c-Fms receptor agonist, such as M-CSF, and iv. optionally a stem cell survival supplement, such as CloneR™ 2. Each of the components i), ii), iii), or iv) may be replaced with a peptide, protein or small molecule, or combinations thereof including a number of each, that would activate the same receptor(s) or intracellular signalling pathway(s).

[0538] The hepatocyte growth factor (HGF) receptor agonist, such as HGF, may signal by GABI to activate

[0539] PI3K, SHP2, and PLC-y signalling, and may further activate other growth- and / or proliferationpromoting pathways. The HGF receptor agonist, such as HGF, may be provided at a concentration of about 50 ng / ml. The HGF receptor agonist, such as HGF, may be provided at a concentration of at least

[0540] 1 picogram / mL In another embodiment, the HGF receptor agonist, such as HGF, may be provided at a concentration of at least 30 ng / ml. The HGF receptor agonist, such as HGF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the HGF receptor agonist, such as HGF, may be provided at a concentration of from about 20 ng / ml to about

[0541] 100 ng / ml. The HGF receptor agonist, such as HGF, may only be provided to the cells once i.e. on the first day of the aggregation period, and not be provided again for the rest of the aggregation period.

[0542] The IL-6 receptor agonist and / or GP130 agonist, such as oncostatin M (One M, or OSM) may signal to activate the Jak / STAT and Tyk2 pathways. The IL-6 receptor agonist and / or GP130 agonist, such as One

[0543] M, may be provided at a concentration of about 30 ng / ml. The IL IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of at least 15 ng / ml. The IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided at a concentration of from about 10 ng / ml to about 80 ng / ml. The IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided to the cells on the first day of the aggregation period, and may also be provided again during the aggregation period. For example, in an embodiment comprising mLAs, the IL-6 receptor agonist and / or GP130 agonist, such as One M, may be provided on the first day of the aggregation period (day = DO), and the assembloid aggregation medium may then be refreshed after 5 days of culture (D5), then on refreshing the assembloid aggregation medium the IL-

[0544] 6 receptor agonist and / or GP130 agonist, such as One M, may be provided in the new assembloid aggregation medium. Optionally, and preferably, D5 coincides with the start of the second aggregation period. The c-Fms receptor agonist may signal via the c-Fms receptor to activate ERK and AKT signalling. The c-Fms receptor agonist may comprise M-CSF (Macrophage Colony-Stimulating Factor). Alternatively, the c-Fms receptor agonist may comprise an antibody such as Eflapegrastim. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of about 80 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 1 picogram / ml. In another embodiment, the c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of at least 40 ng / ml. The c-Fms receptor agonist, such as M-CSF, may be provided at a concentration of from about 1 picogram / ml to about 1 g / ml. In another embodiment, the c-Fms receptor agonist, such as M-

[0545] CSF, may be provided at a concentration of from about 40 ng / ml to about 160 ng / ml. The c-Fms receptor agonist, such as M-CSF, may only be provided to the cells once i.e. on the first day of the aggregation period, and not be provided again for the rest of the aggregation period.

[0546] The stem cell survival supplement, such as CloneR™ 2 (STEMCELL Technologies) or an equivalent, may be a combination of compounds that is known to promote cell survival in stem cells such as hiPSCs.

[0547] The stem cell survival supplement may comprise a ROCKi, such as Y-27632. The stem cell survival supplement, such as CloneR™ 2, may additionally increase the cloning efficiency. The stem cell survival supplement, such as CloneR™ 2, may promote cell survival specifically under high-stress conditions, including seeding at low or high densities, for example it may facilitate the survival and expansion of clonal lines generated by low-density seeding. The stem cell survival supplement, such as CloneR™ 2, may increase the survival of cells under stressful conditions, such as after thawing, or when creating monolayers. The stem cell survival supplement, such as CloneR™ 2, may only be provided to the cells once i.e. on the first day of the aggregation period, and not be provided again for the rest of the aggregation period.

[0548] Methods of Differentiation

[0549] The differentiation of hiPSCs that are cultured with the composition may be achieved via a variety of methods. In particular, in some embodiments, particular differentiation methods will result in the differentiation of the hiPSCs that are cultured with the composition into particular cell types.

[0550] In some embodiments, the differentiation method may encourage differentiation of hiPSCs into mesodermal progenitor cells. In these embodiments additional differentiation methods may encourage further differentiation of mesodermal progenitor cells into later mesodermal-lineage cell types, such as hemogenic endothelial cells, hematopoietic progenitor cells, T cells, unpolarised (i.e. naive) macrophages, Ml macrophages, M2 macrophages, immature dendritic cells, mature dendritic cells, fibroblast-like mesodermal progenitor cells, endothelial cells, and cardiomyocytes.

[0551] In other embodiments, the differentiation method may encourage differentiation of hiPSCs into definitive endodermal cells. In these embodiments additional differentiation methods may allow further differentiation of definitive endodermal cells into later endodermal-lineage cell types, such as anterior foregut endodermal cells, posterior gut tube cells, intestinal epithelial cells, hepatoblasts, hepatic-like cells, type II alveolar epithelial cells.

[0552] According to another aspect of the invention, there is provided a method for differentiating induced pluripotent stem cells (iPSCs) into mesodermal progenitor cells, the method comprising:

[0553] 1) providing induced pluripotent stem cells (iPSCs);

[0554] 2) inducing differentiation of the iPSCs into mesoderm by culturing the iPSCs in a mesoderm induction media according to the invention; and

[0555] 3) optionally further differentiating the mesodermal progenitor cells into a different cell type.

[0556] Step 3) may comprise the further differentiation of the mesodermal progenitor cells into hemogenic endothelium, endothelial cells, or fibroblasts. In one embodiment, Step 3) may comprise the further differentiation of the mesodermal progenitor cells into hemogenic endothelium, hematopoietic cells, endothelial cells, fibroblasts, dendritic cells, monocytes, or macrophages. The step 3) differentiation of the mesodermal progenitor cells into the above-mentioned different cell types may be direct, or via one or more intermediate cell types and / or steps.

[0557] Differentiation to Endothelial cells

[0558] In an embodiment, wherein step 3) comprises the further differentiation of the mesodermal progenitor cells into endothelial cells, step 3) may comprise:

[0559] 3) incubating the mesoderm progenitor cells in an endothelial cell induction medium according to the invention.

[0560] Step 3) may be for a period until endothelial cells are formed. The period may be until at least 50% of the cells are endothelial cells. In one embodiment, the period may be until at least 80% of the cells are endothelial cells. In another embodiment, the period may be until 100% of the cells are endothelial cells. Step 3) may be for a period of at least 3 days. Step 3) may be for a period of about 3-21 days.

[0561] Step 3) may be for a period of about 6-8 days. In one embodiment, step 3) is for a period of about 7 days.

[0562] Differentiation to Fibroblasts

[0563] In an embodiment, wherein step 3) comprises the further differentiation of the mesodermal progenitor cells into fibroblasts, step 3) may comprise:

[0564] 3) incubating the mesoderm progenitor cells in a fibroblast induction medium according to the invention.

[0565] Step 3) may be for a period until fibroblasts are formed. The period may be until at least 50% of the cells are fibroblasts. The period may be until at least 80% of the cells are fibroblasts. Preferably, the period may be until 100% of the cells are fibroblasts. Step 3) may be for a period of at least 2 days.

[0566] Step 3) may be for a period of about 2-28 days. Step 3) may be for a period of about 8-10 days. In one embodiment, step 3) is for a period of about 9 days.

[0567] Differentiation to Hemogenic Endothelium

[0568] In an embodiment, wherein step 3) comprises the further differentiation of the mesodermal progenitor cells into hemogenic endothelium, step 3) may comprise:

[0569] 3) incubating the mesoderm progenitor cells in a hemogenic endothelium induction medium according to the invention; and

[0570] 4) optionally further differentiating the hemogenic endothelium to form a different cell type.

[0571] Step 3) may be for a period until hemogenic endothelium cells are formed. The period may be until at least 50% of the cells are hemogenic endothelium cells. The period may be until at least 80% of the cells are hemogenic endothelium cells. Preferably, the period may be until 100% of the cells are hemogenic endothelium cells. The period may be at least 2 days. Step 3) may be for a period of about

[0572] 2-28 days. The period may be 2-4 days, preferably about 3 days. The skilled person will be familiar with techniques to determine the formation of hemogenic endothelium cells.

[0573] Step 4) may comprise the further differentiation of the hemogenic endothelium into hematopoietic cells. In one embodiment, Step 4) may comprise the further differentiation of the hemogenic endothelium into hematopoietic cells; monocytes, or macrophages. The macrophages may be polarized. The macrophages may be M0, Ml or M2, such as M2a, M2b, M2c, or M2d, subtypes.

[0574] In another embodiment, Step 4) may comprise the further differentiation of the hemogenic endothelium into hematopoietic cells; monocytes, or dendritic cells. The dendritic cells may be immature dendritic cells or mature dendritic cells.

[0575] The step 4) differentiation of the hemogenic endothelium into the above-mentioned different cell types may be direct, or via one or more intermediate cell types and / or steps.

[0576] Differentiation to Hematopoietic cells

[0577] In an embodiment, wherein step 4) comprises the further differentiation of the hemogenic endothelium into hematopoietic cells, step 4) may comprise:

[0578] 4) incubating the hemogenic endothelium in a hematopoietic cell induction medium according to the invention; and

[0579] 5) optionally further differentiating the hematopoietic cells to form a different cell type.

[0580] Step 4) may be for a period until hematopoietic cells are formed. The period may be until at least 50% of the cells are hematopoietic cells. The period may be until at least 80% of the cells are hematopoietic cells. In another embodiment, the period may be until 100% of the cells are hematopoietic cells. The period may be at least 2 days. In one embodiment, the period of step 4) may be 2-28 days. The period may be 2-4 days, preferably about 3 days.

[0581] Step 5) may comprise the further differentiation of the hematopoietic cells into monocytes. In one embodiment. Step 5) may comprise the further differentiation of the hematopoietic cells into monocytes, or macrophages. The macrophages may be polarized. The macrophages may be M0, Ml or M2, such as M2a, M2b, M2c, or M2d, subtypes. In another embodiment, Step 5) may comprise the further differentiation of the hematopoietic cells into monocytes or dendritic cells. The dendritic cells may be immature dendritic cells or mature dendritic cells.

[0582] The step 5) differentiation of the hematopoietic cells into the above-mentioned different cell types may be direct, or via one or more intermediate cell types and / or steps.

[0583] Differentiation to T cells

[0584] In an embodiment wherein step 5) comprises the further differentiation of the hematopoietic cells into T cells, step 5) may comprise:

[0585] 5) culturing the hematopoietic cells on a layer of feeder cells such as OP9-DL1 cells, while incubating the cells in a T cell induction medium according to the invention.

[0586] The layer of feeder cells may comprise cell types that confer growth and pluripotent characteristics to cells. The layer of feeder cells may comprise stromal cells, primary human dermal fibroblasts, or embryonic fibroblast cells such as primary mouse embryonic fibroblasts. Preferably, the layer of feeder cells comprises or consists of stromal cells, such as human stromal cell lines (e.g. OP9-DL1 cells, a bonemarrow-derived stromal cell line that ectopically expresses the Notch ligand Delta-like 1 (Dill)). Most preferably, the layer of feeder cells comprises or consists of 0P9-DL1 cells.

[0587] The layer of feeder cells, such as OP9-DL1 cells, may be cultured in the vessel destined for the differentiation of hematopoietic cells into T cells according to the invention for an appropriate length of time prior to the commencement of step 5), such as for a length of time that allows the layer of feeder cells, such as 0P9-DL1 cells, to express proteins useful and / or required for the differentiation.

[0588] That is to say, the layer of feeder cells, such as 0P9-DL1 cells, may be prepared for step 5) during step

[0589] 4) (and any other prior steps, as required). The layer of feeder cells, such as OP9-DL1 cells, may be cultured for about 2 days before step 5). The layer of feeder cells, such as OP9-DL1 cells, may be cultured for about 1-4 days before step 5). The layer of feeder cells, such as OP9-DL1 cells, may be cultured for about 1-3 days before step 5). The layer of feeder cells, such as OP9-DL1 cells, may be cultured for about 2-3 days before step 5). The layer of feeder cells, such as OP9-DL1 cells, may be cultured for about 1-2 days before step 5). Preferably, at the time of plating, the layer of feeder cells, such as OP9-DL1 cells, may be plated at a density of about 2,500-10,000 cells / cm2. More preferably, at the time of plating, the layer of feeder cells, such as OP9-DL1 cells, may be plated at a density of about

[0590] 3,000-7,000 cells / cm2. Most preferably, at the time of plating, the layer of feeder cells, such as OP9-

[0591] DL1 cells, may be plated at a density of about 5,000 cells / cm2.

[0592] The T cell induction medium may be changed / refreshed after 12-24 hours. The T cell induction medium may be changed / refreshed after 60-180 hours. The T cell induction medium may be changed / refreshed after 3-8 days. Preferably, the T cell induction medium may be changed / refreshed after 3-7 days. More preferably, the T cell induction medium may be changed / refreshed after 3-6 days.

[0593] Most preferably, the T cell induction medium may be changed / refreshed after about 3 days.

[0594] Step 5) may be for a period until T cells are formed. The period may be until at least 50% of the cells are T cells. The period may be until at least 80% of the cells are T cells. In another embodiment, the period may be until 100% of the cells areT cells. The period may be at least 2 days. In one embodiment, the period of step 5) may be 2-28 days. The period may be 5-14 days, preferably about 12 days.

[0595] Preferably, the period may consist of 12 days, wherein the T cell induction medium is changed / refreshed after the first about 6 days. The person skilled in the art will realise that the identify of the T cells may be assessed by routine methods in the art, such as flow cytometry for e.g. CD45 and / or CD7.

[0596] Differentiation to Monocytes

[0597] In an embodiment wherein step 5) instead comprises the further differentiation of the hematopoietic cells into monocytes, step 5) may comprise:

[0598] 5) incubating the hematopoietic cells in a monocyte induction medium according to the invention; and

[0599] 6) optionally further differentiating the monocytes to form a different cell type.

[0600] Step 5) may be for a period until monocytes are formed. The period may be until at least 50% of the cells are monocytes. The period may be until at least 80% of the cells are monocytes. In another embodiment, the period may be until 100% of the cells are monocytes. The period may be at least 2 days. In one embodiment, the period of step 5) may be 2-28 days. The period may be 5-7 days, preferably about 6 days. In one embodiment, step 6) may comprise the further differentiation of the monocytes into macrophages. The macrophages may be polarized. The macrophages may be MO, Ml or M2, such as

[0601] M2a, M2b, M2c, or M2d, subtypes.

[0602] In another embodiment, step 6) may comprise the further differentiation of the monocytes into dendritic cells. The dendritic cells may be immature dendritic cells or mature dendritic cells.

[0603] Differentiation to Macrophages

[0604] In an embodiment wherein step 5) comprises the further differentiation of the monocytes into macrophages, step 5) may comprise:

[0605] 6) incubating the monocytes in a macrophage induction medium according to the invention; and

[0606] 7) optionally polarising the macrophages.

[0607] Step 6) may be for a period until macrophages are formed. The period may be until at least 50% of the cells are macrophages. The period may be until at least 80% of the cells are macrophages. In another embodiment, the period may be until 100% of the cells are macrophages. The period may be at least

[0608] 2 days. In one embodiment, the period of step 6) may be 2-28 days. The period may be 3-5 days, preferably about 4 days.

[0609] In one embodiment, step 7) may comprise polarization of the macrophages, for example into Ml or

[0610] M2, such as M2a, M2b, M2c, or M2d, subtypes.

[0611] Macrophage polarization

[0612] In an embodiment wherein step 7) comprises the polarisation of the macrophages, step 7) may comprise:

[0613] 7) incubating the macrophages in a polarisation medium MO, Ml or M2 according to the invention. Step 7) may be for a period until the macrophages are polarised. The period may be until at least 50% of the macrophages are polarised. The period may be until at least 80% of the macrophages are polarised. In another embodiment, the period may be until 100% of the macrophages are polarised.

[0614] The period may be at least 2 hours. The period may be at least 1 day. The period may be for 2-28 hours.

[0615] The period may be 1-3 days, preferably about 2 days.

[0616] The skilled person will recognise that macrophages exposed to LPS and INF-y may develop a clear morphological change i.e. they may become elongated and spindle-like, which may be concurrent with a change to a CDllb+ / CD80+ / CD160- Ml profile. Conversely treatment with IL-4 may induce a morphological change to a more rounded shape and may be concurrent with a change to a

[0617] CDllb+ / CD80- / CD160+ M2 profile. The skilled person will recognise that it may be possible, and advantageous, to assess the efficiency of M<t> polarization by performing flow cytometry using CDllb,

[0618] CD80 and CD86 after step 7).

[0619] Differentiation to Immature dendritic cells

[0620] In an embodiment wherein step 6) comprises the further differentiation of the monocytes into immature dendritic cells, step 6) may comprise:

[0621] 6) incubating the monocytes in an immature dendritic cell induction medium according to the invention; and

[0622] 7) optionally further differentiating the immature dendritic cells to form a different cell type.

[0623] Step 6) may be for a period until immature dendritic cells are formed. The period may be until at least

[0624] 50% of the cells are immature dendritic cells. The period may be until at least 80% of the cells are immature dendritic cells. In another embodiment, the period may be until 100% of the cells are immature dendritic cells. The period may be at least 2 hours. The period may be at least 5 days. The period may be for 2-168 hours. In another embodiment, the period may be for 2-28 hours. The period may be 5-7 days, preferably about 6 days.

[0625] In one embodiment, step 7) may comprise differentiating the immature dendritic cells into mature dendritic cells (i.e. maturing the dendritic cells).

[0626] Differentiation to Mature dendritic cells In an embodiment wherein step 7) comprises the further differentiation of the immature dendritic cells into mature dendritic cells, step 7) may comprise:

[0627] 7) incubating the immature dendritic cells in a mature dendritic cell induction medium according to the invention.

[0628] Step 7) may be for a period until mature dendritic cells are formed. The period may be until at least

[0629] 50% of the cells are mature dendritic cells. The period may be until at least 80% of the cells are mature dendritic cells. In one embodiment, the period may be until 100% of the cells are mature dendritic cells. The period may be at least 2 hours. The period may be at least 1 day. The period may be 2-28 hours. In another embodiment, the period may be 2-72 hours. The period may be 1-3 days, preferably about 2 days.

[0630] Differentiation of iPSCs into Endoderm

[0631] According to another aspect of the invention, there is provided a method for differentiating induced pluripotent stem cells (iPSCs) into endoderm, the method comprising:

[0632] 1) providing induced pluripotent stem cells (iPSCs);

[0633] 2) inducing differentiation of the iPSCs into mesoderm by culturing the iPSCs in an endoderm induction media according to the invention; and

[0634] 3) optionally further differentiating the mesodermal progenitor cells into a different cell type.

[0635] Step 2) may be for a period until endoderm cells are formed. The period may be until at least 50% of the cells are endoderm cells. The period may be until at least 80% of the cells are endoderm cells. In one embodiment, the period may be until 100% of the cells are endoderm cells. The period may be at least 2 hours. The period may be at least 2 days. The period may be 2-96 hours. In another embodiment, the period may be 48-96 hours. The period may be 2-6 days, preferably about 2-4 days.

[0636] Step 3) may comprise the further differentiation of the endoderm into anterior foregut endoderm, lung progenitor cells, or distal lung cells, such as alveolar epithelial type II cells. The step 3) differentiation of the endoderm cells into the above-mentioned different cell types may be direct, or via one or more intermediate cell types and / or steps. Differentiation to anterior foregut endoderm

[0637] In an embodiment, wherein step 3) comprises the further differentiation of the endoderm into anterior foregut endoderm, step 3) may comprise:

[0638] 3) incubating the endoderm in an anterior foregut endoderm induction medium according to the invention; and

[0639] 4) optionally further differentiating the anterior foregut endoderm cells into a different cell type.

[0640] Step 3) may be for a period until anterior foregut endoderm cells are formed. The period may be until at least 50% of the cells are anterior foregut endoderm cells. The period may be until at least 80% of the cells are anterior foregut endoderm cells. In one embodiment, the period may be until 100% of the cells are anterior foregut endoderm cells. The period may be at least 2 hours. The period may be at least 1 day. The period may be 2-72 hours. In another embodiment, the period may be 48-72 hours.

[0641] The period may be 1-3 days, preferably about 3 days.

[0642] Step 4) may comprise the further differentiation of the anterior foregut endoderm cells into lung progenitor cells, or distal lung cells, such as alveolar epithelial type II cells. The step 4) differentiation of the anterior foregut endoderm cells into the above-mentioned different cell types may be direct, or via one or more intermediate cell types and / or steps.

[0643] Differentiation to lung progenitor cells

[0644] In an embodiment, wherein step 4) comprises the further differentiation of the anterior foregut endoderm cells into lung progenitor cells, step 4) may comprise:

[0645] 4) incubating the anterior foregut endoderm cells in a lung progenitor cell induction medium according to the invention; and

[0646] 5) optionally further differentiating the lung progenitor cells into a different cell type.

[0647] Step 4) may be for a period until lung progenitor cells are formed. The period may be until at least 10% of the cells are lung progenitor cells. In another embodiment, the period may be until at least 20% of the cells are lung progenitor cells. The period may be until at least 50% of the cells are lung progenitor cells. In one embodiment, the period may be until at least 80% of the cells are lung progenitor cells. In another embodiment, the period may be until 100% of the cells are lung progenitor cells. The period may be at least 3 days. The period may be at least 5 days. In one embodiment, the period may be 3-

[0648] 30 days. The period may be 5-7 days, preferably about 6 days.

[0649] Step 5) may comprise the further differentiation of the lung progenitor cells into distal lung cells, such as alveolar epithelial type II cells.

[0650] Differentiation to distal lung cells, such as alveolar epithelial type II cells

[0651] In an embodiment, wherein step 5) comprises the further differentiation of the lung progenitor cells into distal lung cells, such as alveolar epithelial type II cells, step 5) may comprise:

[0652] 5) incubating the lung progenitor cells in a distal lung induction medium according to the invention.

[0653] Step 5) may be for a period until distal lung cells, such as alveolar epithelial type II cells, are formed.

[0654] The period may be until at least 30% of the cells are distal lung cells. In another embodiment, the period may be until at least 50% of the cells are distal lung cells. In one embodiment, the period may be until at least 80% of the cells are distal lung cells. In another embodiment, the period may be until

[0655] 90% of the cells are distal lung cells. In another embodiment, the period may be until 100% of the cells are distal lung cells.

[0656] Differentiation to foregut endoderm

[0657] In an embodiment, wherein step 3) comprises the further differentiation of the endoderm into foregut endoderm, step 3) may comprise:

[0658] 3) incubating the endoderm in a foregut induction medium according to the invention; and

[0659] 4) optionally further differentiating the foregut endoderm cells into a different cell type.

[0660] Step 3) may be for a period until foregut endoderm cells are formed. The period may be until at least

[0661] 50% of the cells are foregut endoderm cells. The period may be until at least 80% of the cells are foregut endoderm cells. In one embodiment, the period may be until 100% of the cells are foregut endoderm cells. The period may be at least 2 hours. The period may be at least 1 day. The period may be 2-72 hours. In another embodiment, the period may be 48-72 hours. The period may be 1-3 days, preferably about 2 days.

[0662] The foregut induction medium may be refreshed every about 24-48 hours during step 3). Preferably, the foregut induction medium is refreshed every about 24 hours during step 3).

[0663] Step 4) may comprise the further differentiation of the foregut endoderm cells into hepatic endoderm cells, or hepatocytes. The step 4) differentiation of the foregut endoderm cells into the abovementioned different cell types may be direct, or via one or more intermediate cell types and / or steps.

[0664] Differentiation to hepatic endoderm

[0665] In an embodiment, wherein step 4) comprises the further differentiation of the foregut endoderm cells into hepatic endoderm cells, step 4) may comprise:

[0666] 4) incubating the foregut endoderm cells in a hepatic endoderm induction medium according to the invention; and

[0667] 5) optionally further differentiating the hepatic endoderm cells into a different cell type.

[0668] Step 4) may be for a period until hepatic endoderm cells are formed. The period may be until at least

[0669] 10% of the cells are hepatic endoderm cells. In another embodiment, the period may be until at least

[0670] 20% of the cells are hepatic endoderm cells. The period may be until at least 50% of the cells are hepatic endoderm cells. In one embodiment, the period may be until at least 80% of the cells are hepatic endoderm cells. In another embodiment, the period may be until 100% of the cells are hepatic endoderm cells. The period may be at least 3 days. The period may be at least 4 days. The period may be at least 5 days. In one embodiment, the period may be 3-10 days. The period may be 3-7 days, preferably about 4 days.

[0671] The foregut induction medium may be refreshed every about 24-72 hours during step 4). Preferably, the foregut induction medium is refreshed every about 48 hours during step 4).

[0672] Step 5) may comprise the further differentiation of the hepatic endoderm cells into hepatocytes.

[0673] Differentiation to hepatocytes In an embodiment, wherein step 5) comprises the further differentiation of the hepatic endoderm cells into hepatocytes (also known as hepatic-like-cells, HLCs), step 5) may comprise:

[0674] 5) incubating the hepatic endoderm cells in a hepatic maturation medium according to the invention.

[0675] Step 5) may be for a period until HLCs are formed. The period may be until at least 30% of the cells are

[0676] HLCs. In another embodiment, the period may be until at least 50% of the cells are HLCs. In one embodiment, the period may be until at least 80% of the cells are HLCs. In another embodiment, the period may be until 90% of the cells are HLCs. In another embodiment, the period may be until 100% of the cells are H LCs. The period may be at least 5 days. The period may be at least 10 days. The period may be at least 14 days. In one embodiment, the period may be 5-15 days. Preferably, the period may be about 10-14 days.

[0677] The hepatic maturation medium may be refreshed every about 24-72 hours during step 5). Preferably, the hepatic maturation medium is refreshed every about 48 hours during step 5).

[0678] Differentiation to cardiomyocytes

[0679] In other embodiments, the differentiation method may encourage differentiation of hiPSCs into cardiomyocytes (CMCs), also known as cardiac myocytes.

[0680] Thus, according to another aspect of the invention, there is provided a method for differentiating induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMCs), the method comprising:

[0681] 1) providing induced pluripotent stem cells (iPSCs);

[0682] 2) inducing differentiation of the iPSCs into immature CMCs by culturing the iPSCs in a CMC induction medium according to the invention; and

[0683] 3) optionally further differentiating the immature CMCs cells into mature CMCs by culturing the immature CMCs in a CMC maturation medium.

[0684] Step 2) may be for a period until immature CMCs are formed. The period may be until at least 50% of the cells are immature CMCs. The period may be until at least 80% of the cells are immature CMCs. In one embodiment, the period may be until 100% of the cells are immature CMCs. The period may be at least about 5 days. Preferably, the period may be at least about 6 days. More preferably, the period may be about 7. During this period, the CMC induction medium may be refreshed and / or supplemented with particular supplements according to the invention herein.

[0685] Step 3) may be for a period until mature CMCs are formed. The period may be until at least 50% of the cells are mature CMCs. The period may be until at least 80% of the cells are mature CMCs. In one embodiment, the period may be until 100% of the cells are mature CMCs. The period may be at least about 1 day. Preferably, the period may be at least about 5 days. More preferably, the period may be about 7 days. Most preferably, the period may be about 15 days. During this period, the CMC maturation medium may be refreshed, such as every about 24 hours, or every about 48 hours, or every about 72 hours, or more (i.e. less often).

[0686] Each incubation step of the methods of the invention may comprise the changing of the medium to from one step to another, with the cells remaining in situ. Mediums may or may not be changed / refreshed periodically during the incubation, for example, replaced with the same medium.

[0687] Medium changes may be every 12-72 hours.

[0688] Incubations of the cells may be on standard tissue plates or dishes, such as multi-well-plates.

[0689] Incubation may be carried out under standard conditions, such as standard gas and temperature conditions known to the skilled person. For example, 37°C and 5% CO2.

[0690] Further Aspects

[0691] According to another aspect of the present invention, there is provided the use of the composition according to the invention for differentiating iPSCs, optionally wherein the iPSCs are differentiated into mesodermal progenitor cells.

[0692] According to another aspect of the present invention, there is provided the use of the composition according to the invention for differentiating iPSCs, optionally wherein the iPSCs are differentiated into mesodermal-lineage cell types. Mesodermal-lineage cell types may comprise mesodermal progenitor cells, hemogenic endothelial cells, hematopoietic progenitor cells, T cells, unpolarised (i.e. naive) macrophages, Ml macrophages, M2 macrophages, immature dendritic cells, mature dendritic cells, fibroblast-like mesodermal progenitor cells, endothelial cells, or cardiomyocytes. According to another aspect of the present invention, there is provided the use of the composition according to the invention for differentiating iPSCs, optionally wherein the iPSCs are differentiated into endodermal-lineage cell types. Endodermal lineage cell types may comprise definitive endodermal cells into later endodermal-lineage cell types, such as anterior foregut endodermal cells, posterior gut tube cells, intestinal epithelial cells, hepatoblasts, hepatic-like cells, or type II alveolar epithelial cells.

[0693] As an alternative to human, the cells types described herein may be non-human animal cells, such as non-human mammalian cells. In one embodiment, the hiPSCs discussed herein may be exchanged with iPSCs, which can comprise human and / or non-human iPSCs. In one embodiment, the iPSCs are human.

[0694] In another embodiment, the iPSCs are non-human. The cell types may be mouse, rat, pig, of simian. as well as equivalent primary derived human cells.

[0695] According to another aspect of the present invention, there is provided the use of the composition according to the invention for isolation and / or culture of mammalian cells, such as fibroblasts, macrophages, cardiomyocytes, lung epithelium, or endothelial cell. Such mammalian cells may be isolated from the human or animal body.

[0696] The composition may be used for the primary or fetal cell equivalent to the cells described herein.

[0697] Such cells may be human, or may be non-human mammalian, such as mouse, rat, pig or simian.

[0698] The iPSCs may be human (hiPSCs).

[0699] The Organoids

[0700] In certain embodiments, the cells obtained by the differentiation of hiPSCs according to the present invention may further assemble, or be assembled, into organoids. Organoid assembly may be spontaneous, or promoted by factors such as medium, temperature, pH, scaffolds, other cell types, or a combination thereof. Organoid assembly may be both spontaneous and promoted by any number of these factors.

[0701] The organoids may assemble, or be assembled, in the composition according to the present disclosure.

[0702] Alternatively or additionally, the organoids may assemble, or be assembled, in the presence (at least temporarily) of at least one other composition. The methods of the invention herein may further comprise the formation of an organoid. In another embodiment, the resulting cells of any one of the methods according to the invention may be clustered together in the form of an organoid.

[0703] According to another aspect of the present invention, there is provided an organoid formed by the methods described herein.

[0704] According to another aspect of the present invention, there is provided a disease model, wherein the disease model comprises an organoid according to the invention that has been treated with an agent to induce a disease state in the organoid.

[0705] The disease may be a congenital disease.

[0706] Pathological features of the disease may be studied and / or differences to non-congenital disease controls may be determined.

[0707] In one embodiment, an agent is used to induce a disease state in the organoid. In one embodiment the agent comprises or consists of a growth factor or cytokine. In another embodiment, a disease or a disorder may be induced genetically, such as by genetic modification, genetic overexpression, or by siRNA silencing, or by treatment with other proteins or pharmacological agents.

[0708] The amount of agent and incubation time with the agent may respectively be an amount and time sufficient to cause disorder in the organoid.

[0709] According to another aspect of the present invention, there is provided the use of the disease model according to the invention to identify agents capable of preventing or treating the disease, wherein the organoid is treated with a potential agent before, during or after the organoid is treated with a disease-inducing agent.

[0710] In another embodiment, disease induced by an agent may be prevented by pharmacological or genetic manipulation of the organoid. The use may comprise the determination of whether the treatment by the potential agent has any effect in inhibiting or reducing the development of disease, or the reduction in disease after it has developed.

[0711] According to another aspect of the present invention, there is provided a method of screening for agents capable of preventing or treating a disease, for example using the organoid described herein, wherein the organoid is genetically manipulated or treated with a potential agent before, during or after the organoid is treated to induce disease; and determining if the potential agent or genetic target has any effect in inhibiting or preventing the development of disease in the organoid, or the reduction in disease after it has developed in the organoid.

[0712] The agent to be screened or investigated may be dosed at a physiological relevant amount. The agent to be screened or investigated may be dosed at a therapeutically relevant amount. Combinations of agents may be investigated.

[0713] The determination may be relative to an untreated organoid (i.e. not treated with the potential agent) and / or relative to a control or reference value.

[0714] In one embodiment the agent to be investigated is a small molecule (e.g. less than 900Da), nucleic acid, antibody therapy, cellular therapy, drug compound, metabolite, or peptide. In one embodiment the agent to be investigated is a small molecule (e.g. less than 900Da), nucleic acid, or peptide. The peptide may comprise or consist of an antibody. In another embodiment the agent to be investigated is a genetic manipulation agent, such as siRNA, shRNA, CRISPR-CAS9, lentiviral or retroviral vectors, for example for over expression.

[0715] According to another aspect of the present invention, a method is provided for seeding of the organoids with cells from a donor, and tracking the cells to assay one or more of survival, proliferation and isolation of engrafted cells, for example for downstream functional testing.

[0716] The cells may be tracked by fluorescent markers or tags. Tracking the cells may comprise the use of a fluorescent cell tracking system. Cells may be treated to express fluorescent markers or tags (such as by genetic material-induced overexpression of fluorescently-tagged proteins). Alternatively or additionally, cells may be treated so to render them fluorescent, e.g. by treatment with a fluorescent chemical tag or marker that is incorporated by at least some of the cells, or with a chemical tag or marker that becomes fluorescent after incorporation by the cells. The person skilled in the art will be familiar with many such tagging systems, and will be able to choose one or more according to the requirements of a particular embodiment.

[0717] In one embodiment, the organoids according to the invention may be used to produce a 3D culture model, for example a model resembling a disease or condition.

[0718] According to another aspect of the present invention, there is provided a method for producing a product from the organoids according to the invention, the method comprising the incubation of the organoids in vitro, and harvesting the product produced from the organoids.

[0719] The product may be produced naturally by the organoids or induced. The product may be harvested by separating them from the cells of the organoids. The product may comprise cells, tissue, proteins. nucleic acids or other biological molecules.

[0720] According to another aspect of the present invention, there is provided a method of screening for biomarkers of a disorder in an organ or tissue, the method comprising the monitoring of biomarkers released from the organoids or cells engrafted therein, or biomarkers in tissue or cellular extracts of the organoids, or released in the medium that is and / or has been in contact with the organoids.

[0721] The disorder may be a disease state. The disorder may be an infection, genetic disorder, immunological response, cancer state, biological pathway irregularity, or biochemical irregularity. A disorder may develop, or be induced in the organoid, whereby changes to the biomarker profile may be determined and linked to the disorder.

[0722] The biomarkers may comprise proteins, glycoproteins, glycans, peptides, nucleic acids, or any cellular product which may indicate a disorder of the organoid or engrafted cells therein. The biomarkers may be cell markers, such as surface proteins and / or secretions.

[0723] In another aspect, there is provided a kit comprising one or more, such as all, of the cell culture mediums described herein, which are required to carry out any method disclosed herein.

[0724] One component of the composition may initially be provided in the kit already mixed with at least one other component of the composition, thus advantageously reducing the number of components to be freshly mixed in order to produce the full composition. Component stability may be the discriminating factor in deciding whether a given component may be pre-mixed with other components, or whether it may be more advantageous for the component to be provided in the kit physically separated from the other components.

[0725] In another aspect, there is provided a method of therapy comprising the implantation into an animal

[0726] (e.g. a human) of an organoid or assembloid according to the invention herein.

[0727] In another aspect, there is provided a method of therapy comprising the delivery to an animal (e.g. a human) of a cell obtained by any of the methods according to the invention herein.

[0728] The kit may comprise recombinant growth factors and / or cytokines. The recombinant growth factors and / or cytokines may be provided in combined solutions in the kit, or two, three, four, five, six, seven or more separate solutions in the kit.

[0729] The kit may further comprise a set of instructions. The instructions will enable the reader to perform any method disclosed herein.

[0730] The methods herein may employ any step of any other method recited herein.

[0731] In yet a further aspect, there is provided cells obtained by any method disclosed herein, such as one or more specific cell types that may be harvested from the organoids according to the invention.

[0732] The organoids described herein may be further differentiated into a tissue type, and assembled further into assembloids. Therefore, according to another aspect of the present invention, there is provided an assembloid comprising two or more organoids, wherein at least one organoid is according to the invention herein, or is produced by the method of the invention herein.

[0733] According to another aspect of the present invention, there is provided a method of generating an assembloid, the method comprising incubating two or more different organoids, wherein at least one of the organoids comprises an organoid of the invention described herein, or is produced by the method of the invention. One or more of the organoids may be further differentiated into a tissue type prior to, or after, assembling into an assembloid.

[0734] Inducing aggregation in the organoids or assembloids

[0735] Organoids or assembloids according to the invention may be assembled by culturing different cell types according to the invention onto or into scaffolds, and in cell growth media.

[0736] Any type of cells derived via the methods herein may be assembled into organoids, and / or assembloids. For example, organoids / assembloids may comprise and / or may be derived from a cell population that is reminiscent of animal organs.

[0737] By way of non-limiting example, a multi-tissue liver organoid (mLO) may be assembled from and / or comprise or consist of: macrophages (MCs), dendritic cells (DCs), endothelial cells (VECs), fibroblasts

[0738] (FLCs), and hepatic-like-cells (HLCs), derived according to the invention, and / or as primary (isolated) cells. The ratio of the cells comprising the mLO may be adjusted by seeding a desired number of cells.

[0739] The ratio of the cells comprising the mLO may be adjusted to mimic the composition of animal (such as human) livers. As an example, the ratio of HLCs:VECs:M<t>s:DCs:FLCs at the time of seeding may be about 12:4:3:1:1.

[0740] Other examples of expressly envisaged organoids and assembloids include cardiac, pulmonary, renal, and intestinal organoids and assembloids. The person skilled in the art will easily determine the type of cells to be assembled (and their ratio) according to the methods herein in order to obtain a desired organoid or assembloid.

[0741] The organoid or assembloid may be assembled from a total cell population of about 900,000 cells. The organoid or assembloid may be assembled from a total cell population of about 1 million cells. The organoid or assembloid may be assembled from a total cell population of at least about 100 cells. The organoid or assembloid may be assembled from a total cell population of about 500,000-5,000,000 cells. The organoid or assembloid may be assembled from a total cell population of about 750,000-

[0742] 2,000,000 cells. The person skilled in the art will recognize that the organoid or assembloid may be assembled from a total cell population of a number that is appropriate for the size of the cell culture vessel to be used, and commensurate with the total mass expected from the fully-assembled organoid or assembloid. The organoid (or assembloid), such as a mLO, may be assembled by first culturing the desired cells

[0743] (and / or organoids) in a cell culture plate that is shaped to encourage aggregation by gravity, such as a funnel-shaped microwell dish, a pyramid-shape microwell dish, or a hemisphere-shape microwell dish, or a dish comprising well(s) of any shape that is conducive to the aggregation of solids by gravity action.

[0744] This period of culture is known as the first aggregation period. Alternatively, a dish comprising flatbottomed-wells may be employed if the dish is incubated at a tilted angle, that similarly allows the aggregation of solids by gravity action. The first aggregation period may last for a sufficient amount of time to allow at least 70%, 80%, 90%, or more (such as all) of the cells (and / or organoids) to aggregate.

[0745] The first aggregation period may last for about 1-10 days. The first aggregation period may last for about 2-7 days. The first aggregation period may last for about 3-6 days. The first aggregation period may last for about 5 days. In an embodiment comprising mLOs, this first aggregation period may last for about 5 days.

[0746] After the first aggregation period, the cells (and / or organoids) thus assembled may be transferred onto a cell culture plate that exhibits very low cell-adhesive properties, such as an ultra-low adhesion (ULA) dish. The cells (and / or organoids) may be cultured in this plate for a period of time known as the second aggregation period. The second aggregation period may last for a sufficient amount of time to allow at least 70%, 80%, 90%, or more (such as all) of the cells (and / or organoids) to become an organoid, or an assembloid. The second aggregation period may last for about 1-20 days. The second aggregation period may last for about 5-15 days. The second aggregation period may last for about 9-

[0747] 12 days. The second aggregation period may last for about 11 days. In an embodiment comprising mLOs, this second aggregation period may last for about 11 days.

[0748] Xeno-Free

[0749] In a preferred embodiment, all media and media components / supplements used herein may be xeno- free. In particular, all differentiation methods and steps described herein may be in xeno-free medium.

[0750] Feeder-Free

[0751] In a preferred embodiment, differentiation methods and steps described herein may be feeder-free.

[0752] In particular, feeder cells may bot be used in the methods of the invention, unless specifically required.

[0753] It may be advantageous for the methods herein to not to make use of, or comprise, any other ("feeder") cells in co-culture as a source of growth factors and / or source of conditioned growth medium.

[0754] In a preferred embodiment, the differentiation methods and steps described herein may be feeder- free and xeno-free.

[0755] Definitions

[0756] An "assembloid" is intended to refer to an in vitro model that combines two or more organoids, spheroids, or cultured cell types to recapitulate structural and functional properties of an organ. An assembloid may combine any number of organoids together with primary cells, or with cell lines.

[0757] References to "inhibition" or similar, may comprise a reduction in activity or presence of a molecule or the block of a biological pathway, such as a signaling pathway. The inhibition may be total (i.e. 100%) or at least a substantial inhibition. The inhibition may be partial inhibition. Partial inhibition may comprise significant inhibition in order to affect the desired outcome of the inhibition.

[0758] It is understood that "chemically defined medium" is a growth medium suitable for the in vitro cell culture of human or animal cells in which all of the chemical components are known.

[0759] As used herein, the term "organoid" may be taken to describe a self-renewing 3-dimensional, multilineage cellular structure that resembles a specific organ or tissue. Unlike naturally occurring organs and tissue, organoids are grown ex vivo, in miniature and in multiples, typically forming multiple structures that are a micrometers to millimeters in diameter, such as the size of between 50 pm and 5 mm, or between 800 pm and 2 mm. A key distinguishing feature of organoids is that they are grown reproducibly with multiple replicates, enabling experimental studies. The organoids can contain cell types that are transcriptionally representative of specific tissues or organs.

[0760] The skilled person will recognise that the term "organoid" may alternatively be termed "spheroid" or

[0761] "microtissue".

[0762] The term "xeno-free" means a composition only comprised of human-derived components and does not incorporate foreign species such as bovine or porcine. The skilled person will understand that variants of a given protein or amino acid sequence may be available, where the variant is still functional. Such variants may have 1, 2, 3, 4, 5 or more residue substitutions, deletions, or additions, without eliminating the original / wild-type function. The substitutions may be for similar amino acids having equivalent charge, size or hydrophobicity (e.g. conservative substitutions). Variants may comprise protein sequences having at least 70% sequence identity with the wild-type sequence. In another embodiment, variants may comprise protein sequences having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the wild-type sequence. Additionally or alternatively, variants of the protein may comprise truncations relative to the wild-type.

[0763] Sequence identity may be determined by standard BLAST alignment parameters (provided by http: / / www.ncbi.nlm.nih.gov / ).

[0764] The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention.

[0765] The skilled person will recognise that the compositions, components, and medium according to the present invention may be easily adapted as necessary to various other embodiments, while retaining substantially similar functions and achieving substantially similar results.

[0766] The methods and compositions herein may use the components as listed, or alternative components may be provided. The skilled person will recognise that components can be substituted with the same specified or generic components from different sources / manufacturers or brands. The skilled person will appreciate that the exact content and quantities in such mediums may be varied without affecting the ability of the medium to support a particular cell culture, maturation or differentiation. For example, at least 10% variation in quantities may be tolerated. Furthermore, particular components, agents, substances, inhibitors, growth factors and cytokines may be substituted with another which may perform a similar function.

[0767] In embodiments wherein different components of the composition each comprise overlapping components (e.g. compounds, salts, ions, proteins, peptides, amino acids), the given concentration values are to be understood as separate values. By way of hypothetical example only, in embodiments wherein L-Alanine is comprised in each of the IMDM, the F12, and the NEAA supplement, in concentrations of 0.28 mM, 0.1 mM, and 0.1 mM respectively, and in no other component of the composition of those hypothetical embodiments, then the total concentration of L-Alanine in the composition is the sum of the three, i.e. (0.28 + 0.1 + 0.1) mM = 0.48 mM.

[0768] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

[0769] FIGURES

[0770] Figure 1. Differentiation of hiPSCs to mesoderm in CDUM. (A) schematic showing protocol to generate FLCs, MPs, DCs and VECs. (B) Schematic showing protocol to generate mesoderm from hiPSCs in CDUM. (C) Representative brightfield microscope image of mesoderm cells. (D)

[0771] Representative flow-cytometry of CD140a+ mesoderm cells. (E) Analysis of flow cytometry for

[0772] CD140+ mesoderm, n=3. (F) Representative flow-cytometry profiles for multiple lineage markers on mesodermal cells.

[0773] Figure 2. Differentiation of hiPSCs to VECs in CDUM. (A) Schematic showing protocol to generate VECs from hiPSCs. (B) Representative bright-field microscopy image of VECs. (C)

[0774] Representative flow-cytometry profile of VECs. (D) Analysis of VECs expressing lineage specific markers, n=3. (E) Representative bright-field microscopy image of tube forming assay using

[0775] VECs. (F) Representative immunofluorescent images of HUVECs and hiPSC derived VECs expressing eNOS.

[0776] Figure 3. Differentiation of hiPSCs to FLCs in CDUM. (A) Schematic showing a protocol to generate FLCs from hiPSCs in CDUM. (B) Representative bright-field microscopy images of

[0777] FLCs. (C) Representative flow cytometry profile of primary immortalised fibroblasts expressing

[0778] FSP1. (D) Representative flow-cytometry profiles of hiPSC derived FLCs at different timepoints. (E) Analysis of FSP1 expression in FLCs at different time points, n=3. (F) representative immunofluorescence images of FLCs treated with TGFp. (G) Quantification of SMA expression in TGFP treated FLCs.

[0779] Figure 4. Differentiation of hiPSCs to Monocytes. (A) Schematic showing protocol for generating HE. (B) Representative bright-field microscopy images of HE cells. (C)

[0780] Representative flow-cytometry profile of HE. (D) Analysis of flow-cytometry profile of HE, n=3. (E) Representative flow-cytometry profiles of HE showing expression of lineage specific markers. (F) Schematic showing protocol for generating haematopoietic progenitor cells

[0781] (HPCs). (G) representative bright-field microscopy images of HPCs. (H) Representative flowcytometry profile of HPCs. (I) Analysis of flow-cytometry profiling of HPCs, n=3. (J)

[0782] Representative flow-cytometry profiles of HPCs expressing lineage markers. (K) Schematic showing protocol for generating monocytes. (L) Representative bright-field microscopy images of monocytes. (M) Representative flow-cytometry profile of monocytes. (N) Analysis of flow cytometry profiling of monocytes, n=3. (O) Representative flow-cytometry profiles of monocytes expressing lineage markers.

[0783] Figure 5. Differentiation and characterisation of hiPSC derived macrophages. (A) Schematic showing protocol to generate MPs and DCs from monocytes. (B) Schematic showing protocol for differentiation of MPs from monocytes. (C) Representative bright-field microscopy images of MPs. (D) Representative flow-cytometry profile of MPs. (E) Analysis of flow-cytometry profiling of MPs, n=3. (F) Schematic showing strategy for polarising MPs. (G) Representative bright-field microscopy of polarised MPs. (H) Representative flow cytometry profiles of polarised MPs. (I) Analysis of flow-cytometry profiling of polarised MPs, n=3. (J) Representative immunofluorescent images of (Dil)-labeled acetylated low-density lipoprotein (AcLDL) uptake assay in polarised MPs. (K) Representative flow-cytometry profiling of AcLDL uptake in polarised MPs. (L) Quantification of AcLDL uptake in polarised MPs, n=3. (M) Representative flow-cytometry profiles of AcLDL uptake showing intracellular localisation. (N) Representative image-flow-cytometry showing intracellular localisation of AcLDL. (O) Quantification of AcLDL internalisation signal. (P) Representative bright-field microscopy images of FITC-bead uptake assay. (Q) Representative motion tracing of MPs. (R) Representative quantification of cumulative and total distance travelled by MPs. (S) Comparison of MP differentiation efficiency using three hiPSC lines, n=3.

[0784] Figure 6. Generation of DCs from monocytes. (A) Schematic showing protocol for generating

[0785] DCs. (B) Representative bright-field microscopy images of immature (iDCs) and mature (mDCs)

[0786] DCs. (C) Representative flow-cytometry profiles of mDCs expressing lineage markers. (D)

[0787] Representative flow cytometry profile of mDCs expressing mature DC markers. (E)

[0788] Quantification of flow-cytometry profiling of DCs, n=3. (E) Representative image flow- cytometry of FITC-bead uptake assay. (G) Comparison of DC differentiation efficiency in three independent cell lines, n=3. (H) Comparison of maturation efficiency in three independent cell lines, n=3.

[0789] Figure 7. Differentiation of hiPSCs into multiple cell types using Laminin521. (A) Representative bright-field microscopy images of cells at different stages of differentiation cultured on laminin

[0790] 521. (B) Representative images of MPs cultured of Laminin 521. (C) Representative flow- cytometry profile of polarised MPs. (D) Representative flow-cytometry profiles of polarised

[0791] MPs. (E) Representative bright-field microscopy images of I DCs and mDCs. (F) Representative flow-cytometry profiles of mDCs. (G) Representative bright-field microscopy images of VECs.

[0792] (H) Representative flow-cytometry profiles of VECs. (I) Representative bright-field microscopy images of FLCs. (J) Representative flow-cytometry profile of FLCs expressing FSP1. (K)

[0793] Representative flow-cytometry profile of FLC expressing fibroblast markers.

[0794] Figure 8. Hierarchical Clustering and PCA of hiPSC derived cell types. (A) Hierarchical clustering analysis of all cell types (B) and PCA plot. (C) Hierarchical clustering analysis of MPs and (D)

[0795] PCA plot. ( E) Hierarchical clustering analysis of DCs and (F) PCA plot. (G) Hierarchical clustering analysis of VECs and (H) PCA plot. (I) Hierarchical clustering analysis of FLCs and (J) PCA plot.

[0796] Figure 9. Heat Maps and Volcano Plots. (A) Heat maps of each stage of differentiation compared to hiPSCs. (B) Volcano plot for mesoderm, (C) HECs, (D) HPCs, (E) monocytes, (F)

[0797] MPs MO, (G) MPs Ml, (H) MPs M2, (I) DCs (J) VECs and (K) FLCs.

[0798] Figure 10 (SUPPLEMENTARY FIGURE 1): Screening of chemically-defined, xeno-free medium that supports stem cell derived cell types from BE31 cells. (A) Bright field microscopy images showing differentiation of hiPSCs to HLCs in CDUM. (B) Bright-field microscopy of D15 HLCs.

[0799] (C) Representative flow cytometry for markers of mature HLCs. (D) Analysis of flow cytometry for mature HLCs n=3. (E) and (H) Q-PCR showing mRNA expression of HLC genes. (F) Schematic showing protocol to generate HLCs. (G) Representative flow-cytometry profiles of DE D4 and quantification, n=3. DE = Definitive endoderm, FG = foregut, HE = hepatic endoderm.

[0800] Figure 11. SUPPLEMENTARY VECs FIGURE. Differentiation and characterisation of VECs. (A)

[0801] Representative bright-field microscopy images of tubes formation assay using VECs at different seeding densities. (B) Analysis of flow-cytometry profiling of VECs exposed to different growthfactor compositions, n=3). (C) Analysis of flow-cytometry profiling of VECs exposed to three different methods to induce VECs. (D) Representative bright-field microscopy images of VECs cultured using different growth factor combinations. (E) Representative immunofluorescence images of eNOS expression in VECs cultured in different growth-factor combinations. (F)

[0802] Representative flow-cytometry profiles of VECs cultures in different growth-factor combinates.

[0803] (G) Representative bright-field microscopy images of VECs cultures in different growth factor combinations.

[0804] Figure 12. SUPPLEMENTARY FIBROBLAST FIGURE 3. Optimisation of FLC differentiation. (A) schematic showing screening protocol to generate FLCs. (B) Representative bright-field microscopy images of FLCs cultured with or with adenosine in CDUM. (C) Representative flow profiles of D9 FLCs cultures in CDUM with or without adenosine. (D) Representative flow profiles of Dll FLCs cultures in CDUM with or without adenosine. (E) Representative flow cytometry profiles of D6, D9, and Dll FLCs across an extended panel of cell surface markers.

[0805] Figure 13. SUPPLEMENTARY DC FIGURE 6: Optimisation and characterisation of hiPSC derived

[0806] DCs. (A) Representative bright-field microscopy images of mDCs and various densities and timepoints. (B) Representative flow-cytometry profiles of mDCs at different densities on D20.

[0807] (C) Representative flow-cytometry profiles of mDCs at D21. (D) Representative flow-cytometry profiles of mDCs at D22.

[0808] Figure 14. SUPPLEMENTRY LoPCK: Differentiation of multiple cell types from LoPCK hiPSCs cell line. (A) Schematic showing protocol to generate monocytes. (B) Representative bright-field microscopy images of cells at different timepoints. (C) Representative flow-cytometry profiles of monocytes derived from LoPCK hiPSCs. (D) Schematic for polarising MPs. (E) Representative bright-field microscopy images of polarised MPs. (F) Representative flow cytometry profiles of polarised MPs and quantification, n=3. (G) Schematic of protocol for generating DCs. (H)

[0809] Representative bright-field microscopy images of iDCs and mDCs. (I) Representative flowcytometry profiles of mDCs and quantification, n=3. (J) Representative bright-field microscopy images of FLCs. (K) Representative flow-cytometry profiles of FLCs at different time points, and quantification n=3. (L) Representative bright-field microscopy images of VECs. (M)

[0810] Representative flow-cytometry profiles of VECs (N) and quantification n=3.

[0811] Figure 15. SUPPLEMENTRY MoPCK: Differentiation of multiple cell types from MoPCK hiPSCs cell line. (A) Schematic showing protocol to generate monocytes. (B) Representative bright- field microscopy images of cells at different timepoints. (C) Representative flow-cytometry profiles of monocytes derived from MoPCK hiPSCs. (D) Schematic for polarising MPs. (E)

[0812] Representative bright-field microscopy images of polarised MPs. (F) Representative flow cytometry profiles of polarised MPs and quantification, n=3. (G) Schematic of protocol for generating DCs. (H) Representative bright-field microscopy images of iDCs and mDCs. (I)

[0813] Representative flow-cytometry profiles of mDCs and quantification, n=3. (J) Representative bright-field microscopy images of FLCs. (K) Representative flow-cytometry profiles of FLCs at different time points, and quantification n=3. (L) Representative bright-field microscopy images of VECs. (M) Representative flow-cytometry profiles of VECs (N) and quantification n=3.

[0814] Figure 16. A: Schematic diagram of stepwise protocol to induce hiPSCs differentiation into alveolar epithelial type II cells. B: Schematic diagram of stepwise strategy to induce hiPSCs differentiation into M(D, which takes total of 21 days (from seeding D-2). C: Schematic diagram of stepwise strategy to induce hiPSCs differentiation into DC, which takes total of 23 days (from seeding D-2). D: Schematic diagram of stepwise strategy to induce hiPSCs differentiation into

[0815] ECs, which takes total of 9 days (from seeding D-2). E: Schematic diagram of stepwise strategy to induce hiPSCs differentiation into FBs, which takes total of 11 days (from seeding D-2). F:

[0816] Schematic diagram of stepwise strategy to induce hiPSCs differentiation into hepatocytes (or hepatic-like-cells.

[0817] Figure 17. Differentiation of BE31 hiPSCs to T cells by co-culturing with 0P9-DL1. (A) Schematic of protocol for generating T cells. (B) Representative bright-field microscopy images of T cells.

[0818] (C) Representative flow-cytometry profiles of T cells and quantification, n=2. Scale bars 200 pm.

[0819] Figure 18. Differentiation of hPSCs into cardiac cells. Cell morphology during cardiac differentiation using Hannan's defined medium, starting from two different cell lines (MoPCK and C3G3). By day 10, the cells begin exhibiting contractile movements, typical of cardiomyocytes (CMCs) in culture.

[0820] Summary

[0821] Our understanding of human disease is constrained due to reliance non-human disease models.

[0822] Animal and ex-vivo human disease models have provided important insights, however translational application remains challenging due to species and donor-to-donor variability. Stem-cell-derived tissues are important model platforms enabling fine tuning of genetic, cellular, immune and ECM parameters that mimic normal and diseased tissue. Here we develop a serum and xeno-free platform for production of stem-cell-derived, self-organising 3D multitissue organoids. Populations of naive endothelial, fibroblasts, macrophages and dendritic cells were generated using a unified differentiation platform and when combined with mature, stem-cell-derived parenchymal cells, naive cells adopt tissue specific phenotypes consistent with normal and diseased tissue. Our platform allows precise configuration of immune competent, tissue-specific organoids, that are tuneable to a range of different disease parameters which will facilitate experimental insight into mechanisms of human disease at a resolution previously difficult to achieve using animal and ex-vivo human models.

[0823] INTRODUCTION

[0824] The pathophysiology of human disease is complex, involving genetics, environmental and lifestyle factors[l-4], At the tissue level, disease is driven by the complexity of alterations in cellular metabolism and cell-cell communication, genomic instability, changes in tissue cellular composition, remodelling of tissue architecture and extracellular matrix and changes in vascularisation, inflammation, and immune modulation[5-15].

[0825] Disease models that recapitulate these features has been the domain of animal models[16, 17], While these models have delivered crucial insight into human development and disease, physiological, genetic, immune, and metabolic differences create significant challenges translating animal data to human[18, 19], Human tissue explant models along with primary and immortalised human cell lines offer human specificity however extensive use is limited due to access and supply issues, complex culture requirements, limited experimental life-span and donor-to-donor variability [20-27].

[0826] More recently, development of pluripotent stem cell (PSC)-derived multi-cell-type organoids and microtissues have been described that offer opportunities for more complex disease and developmental modelling

[0028] . Typically these models are created using four different approaches whereby PSCs are; 1) differentiated to the desired cell type and then mixed with primary derived cell types to create an organoid[29, 30]; 2) differentiated towards a primary germ layer that further spontaneously differentiates and self-organises into a 3D organoid [31-34]; 3) co-differentiated into multiple cells types using protocols designed to introduce heterogeneity into the resulting population of cells which then further spontaneously differentiate and self-organise into complex organoids

[0035] ;

[0827] 4) differentiated into specific cell types using separate differentiation platforms and then recombined in 3D allowing self-organisation into organoids[36-38]. An additional methodology, that may combine any of the above approaches involves the assembly of multiple organoids structures into a mixed organoid conformation termed an "assembloid" that self-organises into a complex, multi-region organoid[39-42].

[0828] While each approach produces organoids with variable cellular composition, complexity and functional profiles, each approach creates limitations in the versatility and application of the organoids. Organoids comprised PSC-derived and primary cells will exhibit batch-to-batch variability and may limit inclusion of some immune cell types

[0043] , single progenitor-cell-derived tissues will be deficient in cell types derived from other germ layers rendering full tissue composition incomplete

[0044] , organoids derived from the spontaneous differentiation of multiple germ layers will exhibit variability in the efficiency of germ layer and progenitor cell differentiation at the level of the cell line and protocol used and subsequently the downstream composition of organoids will amplify this variability [45, 46],

[0829] While recombining individual populations of cells, derived from PCS allows for control of individual cell numbers and the cellular composition of microtissues, a limited number of cell types have been recombined as diverse medium and cell signalling requirements for each cell types complicate their eventual co-culture resulting in limited survival and functionality of individual cell types[48J.

[0830] Here we explored these limitations and developed a chemically defined, xeno-free, serum-free platform to generate fibroblasts, endothelial cells, macrophages and dendritic cells from hiPSCs using a single medium formulation (Fig la). The resulting cells display functional profiles consistent with each cell type and all cells remain viable and functional in the same medium formulation. We combined these cells with hiPSC derived parenchymal cells into organoid and microtissues representative of different organs and disease modalities. Our platform allows for simple, reproducible and precise generation of microtissues that are tuneable along cellular, genetic, and environmental variables and contain cells important for modelling multiple modalities of disease progression and importantly supports inclusion of multiple autologous immune cell types. This platform addresses many of the limitations of current approaches to co-culture organoids and microtissues and will facilitate a greater understanding of the role epithelial, immune, stromal, and vascular cells play in progression of disease.

[0831] RESULTS: hiPSC differentiation to a common CD140a+mesoderm progenitor

[0832] During co-culture, cells are more likely to maintain their cellular and functional phenotypes when cultured in a xeno-free, chemically-defined medium that is optimal for all populations of cells. Therefore, we first set out to establish a universal differentiation medium by screening various media that support growth of hiPSC derived cell types such as definitive endoderm, mesoderm, hepatic-like- cells and intestinal organoids[49-54]. No formulations tested were optimal for all cell types and cell lines, however, modification of our previously reported chemically defined medium (CDM) formulation[52, 53], was sufficient to support all cell types screened derived from three independent hiPSC lines.

[0833] Using Chemically Defined Universal Medium (CDUM), we screened growth factors required for mesoderm differentiation

[0049] using CD140a as a readout for differentiation efficiency. In CDUM,

[0834] Activin-A, BMP-4, CHIR99021 and LY294002 were sufficient to induce gastrulation and generate a monolayer of mesodermal cells over 48hrs (Fig lb,c). Although a broad range of initial cell densities delivered CD140a+cells, 750 cells / cm2hiPSCs was optimal for further downstream differentiation and generated mesodermal cells that were >90% CD140a+across all three cell lines tested (Fig ld,e). To determine if our CD140a+mesoderm was already biased towards other cell types we assessed by flow cytometry expression of markers typical of endothelial cells (CD140a, CD31), haematopoietic progenitor cells (CD34, CD43), and monocyte, macrophage and dendritic cells (CD14, CDllb, CDllc).

[0835] Analysis showed D2 mesoderm cells were negative for all monocyte lineage markers, expressed low levels of CD144 and were 99% CD14+consistent with previous reports generating mesodermal progenitor cells (Fig If). Using mesoderm progenitor cells, we then optimised multiple, independent differentiation protocols to generate fibroblast-like cells (FLCs), endothelial cells (ECs), macrophages

[0836] (MPs) and dendritic cells (DCs) for use in a co-culture system.

[0837] Differentiation of CD140a+mesoderm to endothelial cells.

[0838] Endothelial cells (EC) are essential for formation of the vasculature network throughout the body and regulate important functions in health and disease including inflammation and immunity, hemostasis, angiogenesis and ECM remodelling. Starting with CD140a+mesoderm, we screened previously reported inducers of vascular endothelial cells

[0055] for compatibility with CDUM. CD140+cells cultured in a combination of FGF2, VEGF, 8-Bromoadenosine 3', 5'-cyclic monophosphate and isoprenaline quickly established a cobble-stone-like morphology before forming a monolayer of ECs >85%

[0839] CD144+ / CD31+(Fig 2a,b,c,d) with similar results observed in 2 additional cell lines. To further characterise the ECs we sorted CD1447CD31+ECs and performed a tube formation assay and assessed expression of endothelial nitric oxide synthase (eNOS). CD144+ / CD31+ECs formed tubes at all densities tested with numbers of tubes formed, length of individual tubes and area of tubes equivalent to those formed by HUVECs (Fig 2e). Similarly, eNOS expression was similar to that of HUVECS (Fig 2f) confirming CD144+ / CD31+ECs possess functional characteristics of primary ECs. Lastly, ECs showed expression of cytokines following stimulation with TNFa similar to those produced by HUVECs.

[0840] Differentiation of CD140a+mesoderm to fibroblast-like-cells

[0841] Fibroblasts are a heterogenous population of cells found in most tissues and are critical in maintaining tissue structure and function, unsurprisingly, fibroblast dysfunction is central to many diseases. We therefore assessed the potential of our CD140a+mesoderm to differentiate into fibroblast-like cells

[0842] (FLCs) in CDUM. We screened growth factors previously reported to generate FLCs [56, 57] and assessed by flow cytometry expression of multiple fibroblasts markers including CD13, CD44, CD90,

[0843] CD140a, CD166 and FSP1. Culture of CD140a+mesoderm with several combinations of factors was sufficient to induce expression of fibroblasts markers, however, only the combination of insulin, hydrocortisone, BMP4, EGF and isoprenaline was sufficient to generate FLCs that expressed all markers and FSP1 (Fig 3a). After 5 days of culture CD140a+adopted a typical fibroblast-like morphology and could be expanded for >23 days in CDUM however the greatest proportion of FSP1+FLCs occurred at day 11 of culture with >70% of FLCs expressing FSP1+(Fig 3b,c,d,e). To test FLC functionality we treated

[0844] FLCs with ILip to induce myofibroblast differentiation which resulted in increased expression in myofibroblast marker a-SMA (Fig 3f,g). Similarly, when treated with TNFa, FSP1+ fibroblasts responded by secreting a similar array of cytokines and at similar quantities to those expressed by primary dermal and respiratory fibroblasts.

[0845] Differentiation of CD140a+mesoderm to macrophages and dendritic cells.

[0846] Immune cells play essential roles in both health and disease. Innate immunity is orchestrated by several cell types including macrophages (MPs) that phagocytose cellular debris, microbes, and cancer cells. Similarly, dendritic cells (DCs) are a bridge between innate and adaptive immunity whereby they function via capturing, processing and presenting antigens to T-cells. Presence of both cell types are important for the development of a tissue model that can provide insight into the role of immune cells in health and disease. Next, we tested the capacity of CD140a+ mesoderm to produce both immune cell types in CDUM. MPs and DCs are generated from a common myeloid progenitor and we initially set-out to develop a step-wise protocol to generate CD14+monocytes. We screened for growth factors previously reported be sufficient to generate hemogenic endothelial cells

[0058] , and found that stem cell factor (SCF), VEGF, FGF2 and inhibition of TGF0 signalling in CDUMM generated >75% CD144+cells after 3 days of differentiation (Fig 4a,b,c,d). Further characterisation revealed CD144+ cells were also

[0847] CD14", CD140a", CD43", CDllb", and CDllc" (Fig 4e). We then screened for combinations of factors that would drive differentiation of CD144+cells to non-adherent CD43+cells. Four days of culture in CDUM and IL-3, IL-6, TPO, VEGF, FGF2 and SCF was sufficient to generate a population of non-adherent HPCs

[0848] >95% CD43+(Fig 4f,g,h,i) and CD140a", CDllb" and CDllc" (Fig 4j). Further analysis revealed that ~19% of cells were CD144" suggesting partial commitment towards the monocyte lineage (Fig 4j). To drive further CD43+HPCs to CD14+monocytes we treated cells with M-CSF, IL3 and IL6 in CDUM for 6 days which was sufficient to generate >90% CD144" monocytes (Fig 4k,l,m,n,o).

[0849] Further culture of CD144" monocytes in the presence of M-CSF was sufficient to generate >99% CDllb4" that developed a distinctive irregular shape with a granulated cytoplasm as they differentiated (Fig

[0850] 5a,b,c,d,e). To assess their functional profile, we decided to test if MPs could be polarised towards Ml- like and M2-like MPs using CDUM. CDllb+MPs exposed to LPS and INF-y developed a clear morphological change which became elongated and spindle-like, which was concurrent with a change to a CDllb+ / CD807CD160" Ml MP profile (Fig 5f,g,h). Conversely treatment with IL-4 induced a morphological change to a more rounded shape and was concurrent with a change to a CDllb+ / CD80"

[0851] / CD1604" M2 MP profile (Fig 5f,g,h,i). Next, we assessed the phagocytotic activity by culturing CDllb+

[0852] MPs, MO, Ml-like and M2-like with either acetylated-LDL, FITC-labelled beads or heat-shocked GFP expressing CD14+monocytes. Resting and polarised MPs were able to engulf ac-LDL, FITC-labelled beads and heat-shocked cells with most MPs actively scavenging and engulfing debris (Fig 5j,k,l). To determine localisation of engulfed debris we performed imaging flow cytometry which showed clear intracellular localisation of debris (Fig 5m,n,o,p), while cell tracing also demonstrated cell movement over time (Fig 5q,r).

[0853] Culture of CD14+monocytes with IL-4 and GM-CSF was sufficient to generate >99% CDllc+DCs that displayed classical dendrite-like structures and contained large cytoplasmic vesicles. (Fig 6a, b).

[0854] Functional maturity of CDllc4" DCs was assessed by exposing DCs to TNFa and LPS for 48hrs which generated a populations of DCs >97% CD80+ / CD86+and 48% of those cells were HLA-DR+(Fig 6,c,d,e).

[0855] Similar to MPs, we tested phagocytic activity of DCs which showed DCs were able to scavenge debris and imaging flow cytometry confirmed their cytoplasmic localisation (Fig 6f). Similar results were obtained in two additional stem cell lines (Fig 6g, h).

[0856] Xeno-free Generation of FLCs, MPs, DCs, and ECs

[0857] Validation of our chemically-defined, xeno-free medium was completed using Matrigel™ to facilitate attachment of cells to tissue cultures plates. To make the platform completely chemically-defined and xeno-free we coated tissue culture plates with an animal-free, recombinant laminin 521 in place of

[0858] Matrigel™ and repeated all differentiation protocols as optimised previously. All mature cell types, as well as intermediate cells including mesoderm, HECs, HPCs, and monocyte steps were reproduced without any significant difference in cell morphology, gene expression profile, or population dynamics

[0859] (Fig 7a). MPs were generated with similar efficiencies and could be polarised to Ml and M2 as previously described (Fig 7b,c,d) while DCs also retained typical dendrite morphology and matured to

[0860] HLADR positive cells (Fig 7e,f). Finally, VECs (Fig7g,h) and FLCs (Fig 7i,j,k) also showed similar growth characteristics and cell surface marker expression profiles as VEC's and FLC's generated on Matrigel™ coated plates and flasks. mRNA Sequencing reveals naive phenotype of differentiated cells.

[0861] Cell surface markers expression and functional phenotyping confirm the basic identity of cells generated in CDUM. Next, we sought to further characterise and validate each stage of our differentiation platform by sorting each population of cells and then performing bulk mRNA- sequencing. When all cell populations and intermediates were analysed together, hierarchical clustering and PCA analysis showed distinct clusters of cells types and high reproducibility within each cell type generated (Fig 8a, b). Interestingly, Individual differentiation pathways were analysed hiPSCs, mesoderm, HECs and HPCs clustered within their individual cell populations but also formed a larger cluster a stem and progenitor-like cell cluster, while ECs, FLCs, MPs, DCs formed their own cluster of more mature cell types. As each of the mature cell types generated using our platform are known to take on a gene expression profile characteristic of the tissue within which they reside in vivo, we were interested to determine if any of the cell populations generated in vitro using CDUM were more like tissue resident cells of any particular tissue. Comparing gene expression profiles of each cell type against the human cell and tissue atlas further confirmed the expected cell type of each population, but interestingly there were no particular tissue types that any of the ECs, FLCs, MPs and DCs were biased towards and instead hits were across multiple tissue resident cell types. To further assess potential bias of each cell type we performed hierarchical cluster analysis against publicly available data sets for EC's, FLCs, MPs, and DC isolated from multiple tissue types (see Figure 8).

[0862] METHODS:

[0863] Generation of hiPSCs

[0864] BE31, 27F, LoPCK, Clonel4 were all generated using the Cytotune 2.0 reprogramming kit. 27F contains a homozygous SFTPC mutation and BE31 is its isogenic corrected WT control. All cells were maintained in E8 medium

[0059] supplemented with TGF-P (2ng / mL), FGF2 (lOOng / mL) and Pen / Strep. hiPSCs were split at a ratio of 1:20 using TryplE every 4 days with Y-27632 (lOμM) supplemented into E8 medium for 1 day during passaging for maintenance cultures and seeding cells for all differentiation experiments. For regular maintenance and differentiation of all cell lines flasks and plates were coated with Matrigel at 34.5ug / cm2. For a completely animal and xeno-free culture system cells were cultured in tissue culture plates and flasks coated with recombinant Laminin-Ill or Laminin-121 purchased from Biolamina.

[0865] Differentiation of hiPSCs to mesodermal progenitor cells

[0866] To generate mesoderm progenitor cells, hiPSCs were plated at a density of 750 cell / cm2on tissueculture plates and dishes using TripLE. Y-27632 (lOμM) was added to E8 medium for 24 hrs with E8 changed after 24hrs to complete 48 hrs of culture in E8 medium prior to initiating mesoderm differentiation. To initiate mesoderm differentiation cells were cultured for 48 hrs in CDUM containing

[0867] BMP4 (50ng / mL), LY294002 (IDμM), Activin-A (15ng / mL), CHIR99021 (5μM). Activin-A and CHIR99021 were added on DI only. Efficiency of differentiation was assessed by CD140a expression by flow cytometry with >80% expected to be CD140+to move to subsequent differentiation steps.

[0868] Differentiation of hiPSCs to Macrophage cells

[0869] Mesodermal progenitor cells were differentiated into hemogenic endothelial cells using CDUM for 3 days containing VEGF (50ng / mL), FGF2 (50ng / mL), SB431542 (10μM) and SCF (50ng / mL). Medium was changed daily for 3 days. Hematopoietic progenitor cells were generated by culturing cells in CDUM supplemented with VEGF (50ng / mL), FGF2 (50ng / mL), SCF (50ng / mL), IL-6 (lOng / mL), IL-3 (50ng / mL),

[0870] TPO (50ng / mL) for 4 days changing medium every 48hrs. Monocytes were then generated by culturing cells in CDUM supplemented with IL-3 (lOng / mL), IL-6 (50ng / mL), and M-CSF (80ng / mL) for 6 days changing medium every 72hrs.

[0871] Polarisation of Macrophages

[0872] Macrophages were polarised to Ml-like using CDUM supplemented with INF-y (20ng / mL) and LPS

[0873] (IDOng / mL) for 2 days without medium change. To polarise to M2-like macrophages were cultured in

[0874] CDUM supplemented with IL-4 (20ng / mL) for 2 days without medium change.

[0875] Differentiation of hiPSCs to Dendritic cells

[0876] Dendritic cells were generated by culturing haemopoietic progenitors cells in CDUM supplemented with GM-SCF (50ng / mL) and IL-4 (20ng / mL) for 6 days changing medium every 72 hrs. Immature DCs were matured in CDUM supplemented with TN Fa (lOOng / mL) and LPS (lOOng / mL) for 2 days without any medium change. Differentiation of hiPSCs to Fibroblast-like-cells

[0877] To generate fibroblast like cells mesodermal progenitor cells were cultured in CDUM supplemented with EGF (lOng / mL), Insulin (5ug / mL), BMP4 (5ng / mL), Isoprenaline (2ug / mL), and Hydrocortisone

[0878] (0.5ug / mL) for 9 days with daily medium changes.

[0879] Differentiation of hiPSCs to Endothelial Cells

[0880] To generate endothelial cells mesodermal progenitor cells were cultured in CDUM supplemented with

[0881] VEGF (300ng / mL), FGF2 (200ng / mL) and 8-bromo-cAMP (InM) for 7 days with medium changes every

[0882] 72 hrs.

[0883] Differentiation of hiPSCs to Definitive Endoderm

[0884] To generate definitive endoderm hiPSCs were plated at a density of 20,000 cells / cm2on tissue culture plates and dishes using TrypLE and cultured for 48hrs in E8 medium supplemented with Y-27632

[0885] (10μM) for the first 24 hrs. To initiate differentiation cells were cultured in CDEM supplemented with

[0886] Activin-A, CHIR99021 and LY294002 for 4 days with daily medium changes. CHIR99021 was removed after 24hrs culture. Flow cytometry was used to assess differentiation efficiency with the expectation that cell would be >90% CXCR47cKIT+by day 4.

[0887] Differentiation of hiPSCs to Type II Alveoli Epithelial Cells

[0888] Differentiation of hiPSCs to Hepatic-like-cells

[0889] To generate hepatic-like cells, definitive endoderm was cultured in CDEM supplemented with Activin-

[0890] A to generate foregut stem cell as previously described. Foregut endoderm was then differentiated in

[0891] CDEM in BMP4 and SB431542 to generate hepatoblasts and then matured in CDEM supplemented with HGF and OSM[50, 54], Medium was changed daily up to hepatic maturation when medium was changed every 48hrs.

[0892] Differentiation of hiPSCs to Intestinal Epithelial Cells

[0893] To generate intestinal epithelium, definitive endoderm was posteriorized into posterior gut tube using

[0894] CDEM supplemented with CHIR99021 for 4 days with daily medium changes as previously described.

[0895] Cells wells were then transferred to 3D culture to generate intestinal organoids as previously described

[0050] . ABBREVIATIONS

[0896] CDEM - Chemically Defined Endoderm Medium

[0897] CDUM - Chemically Defined Universal Medium

[0898] ECM - Extracellular Matrix

[0899] FLCs - Fibroblasts-like cells

[0900] HLCs - Hepatic-like Cells

[0901] MPs - Macrophages

[0902] DCs - Dendritic Cells

[0903] VECs - Vascular Endothelial Cells

[0904] HECs - Hemogenic Endothelial Cells

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[0992] Example 2 - Differentiation of hPSCs to alveolar epithelial type II cells

[0993] MATERIALS

[0994] Reagents

[0995] Dulbecco's Modified Eagle Medium (Corning, cat. no. 10-092-CM)

[0996] Recombinant human insulin (Sigma-Aldrich, cat. no. 11376497001)

[0997] • Transferrin (Sigma, T3705)

[0998] Small molecule mix

[0999] Penicillin / Streptomycin (Gibco cat no 15070-063)

[1000] Iscove's Modified Dulbecco's Medium (IMDM; Thermo Fisher Scientific, cat. no. 12440053)

[1001] Ham's F12 medium (Corning, cat. no. 10-080-CV)

[1002] Polyvinyl Alcohol (Sigma-Aldrich, cat no P1836-250G)

[1003] ITSX (Gibco cat no 51500-056)

[1004] Lipids (Gibco cat no 11905031)

[1005] 1-Thioglycerol (MTG; Sigma-Aldrich, cat. no. M6145)

[1006] • Ascorbic acid (Sigma-Aldrich, cat. no. A4544)

[1007] Non-essential Amino Acids (lOOx) (Gibco cat no 11140-035)

[1008] GlutaMAX (lOOx; Thermo Fisher Scientific, cat. no. 35050061)

[1009] • Y-27632 (Selleckchem, S1049)

[1010] CHIR99021 (Sigma, SML1046)

[1011] • Activin A (Peprotech, #120-14P-500)

[1012] LY294002 (Selleckchem, S1105)

[1013] SB431542 (Tocris, 1614)

[1014] Dorsomorphin (Tocris, 3093)

[1015] • Wnt3A (R&D Systems, 5036-WN)

[1016] R-Spondinl (Peprotech, 120-38-250UG)

[1017] BMP4 (Peprotech, 120-05-100)

[1018] Retinoic Acid (Sigma, R2625)

[1019] FGF10 (Peprotech, 100-26) FGF7 (Peprotech, 100-19)

[1020] IBMX (Sigma, 15879)

[1021] 8-bro cAMP (Sigma, B7880)

[1022] Dexamethasone (Sigma, D4902)

[1023] • TrypLE Express Enzyme (Thermo Fisher Scientific, cat. no. 12604021)

[1024] Dulbecco's Phosphate Buffer Solution (Thermo Fisher Scientific, cat. no. 14190144)

[1025] Nunc Tissue culture-treated T25 flasks (Thermo Fisher Scientific, cat. no. 156367)

[1026] Nunc Tissue culture-treated 12-well and 48-well Multidishes (Thermo Fisher Scientific, cat. no.

[1027] 150628 and 150687)

[1028] Disposable sterile filter syringe (50 ml; BD Plastipak, cat. no. 300866)

[1029] Disposable Ministart sterile syringe filter (0.22 pl; Sartorius, cat. no,16532-K)

[1030] Snaplock Microcentrifuge tubes (1.5 ml; Axygen, cat. no. MCT-150-A)

[1031] CloneR2 (STEMCELL TECHNOLOGIES, 100-0691)

[1032] • Gentle Cell Dissociation Reagent (STEMCELL TECHNOLOGIES, 07174)

[1033]

[1034]  Chemically defined, serum-free differentiation of human pluripotent stem cells to type II alveolar epithelial cells

[1035] Procedure

[1036] Preparation of hPSCs for differentiation

[1037] 1. After thawing, passage hPSCs at least twice before starting differentiation. Begin differentiation when cells are at 70-80% confluency.

[1038] 2. Prepare a Matrigel-coated 12-well plate 1 day prior to seeding cells for differentiation.

[1039] 3. Prepare homebrew E8 medium + Y-27632 (10 μM) (reagent setup) (for a 12-well plate, use 1 ml per well for plating).

[1040] 4. Take Matrigel-coated plate from the incubator, aspirate the DMEM / F12, and add 1 ml per well of DPBS.

[1041] 5. Using a P1000 rinse each well by washing the DPBS across the surface 5 times, being careful not to scratch the Matrigel layer.

[1042] 6. Return the 12-well plate with DPBS to the incubator to allow it to warm to 37 °C.

[1043] 7. Aspirate medium from the hPSC culture, wash once with 5 mL DPBS and add 2 ml TrypLE per

[1044] T25 flask. Incubate at 37 °C for 4 min.

[1045] 8. Check for detachment of hPSCs from the Matrigel-coated flask. At this stage cells should be floating, and colonies should be dispersed.

[1046] 9. Add 2 ml DMEM to neutralise the TrypLE and collect cells by gently washing across the surface of the flask.

[1047] 10. Transfer the cells to a 15 ml falcon tube.

[1048] 11. Centrifuge cells for 4 min at 300 g.

[1049] 12. Aspirate the supernatant and gently resuspend the cell pellet in 4 ml of warm DMEM.

[1050] 13. Immediately take 10 μl of cell suspension and place it in a haemocytometer for cell counting.

[1051] 14. Calculate the total number of cells required for seeding using the calculation below. Plate between 20,000 cells / cm2per well. Note this density is very confluent. Lower densities still specify a >99% CXCR4 / CKIT double-positive population, but a higher density leads to a more stable differentiation.

[1052] (Number of wells x surface area of well) x number of cells required per cm2= total number of cells required

[1053] 15. Resuspend the desired volume of cell suspension in E8 + Y-27632 (10 μM) medium.

[1054] 16. Aspirate the DPBS from the 12-well plate and add 1 ml of cell suspension per well of the

[1055] Matrigel-coated 12-well plate. 17. Gently rock the plate from side to side and forward and backward to ensure even distribution of cells and incubate at 37 °C overnight.

[1056] 18. Following 24 h from plating, aspirate the medium and add 500 ul of E8 without Y-27632 per well. Incubate the cells at 37 °C for 24 h.

[1057] Specification of definitive endoderm (Day 0 - 4)

[1058] For all steps of definitive endoderm induction, use CDEM basal medium.

[1059] 19. Aspirate the medium and add 500 μl per well of CDEM + CHIR-99021 (3 μM), Activin A (100 ng / ml) and LY294002 (10 uM). Incubate at 37 °C for 24 h.

[1060] 20. Aspirate the medium and add 500 μl per well of CDEM + Activin A (100 ng / ml) and LY294002

[1061] (lOuM). Incubate at 37 °C for 24 h.

[1062] 21. Aspirate the medium and add 500 μl per well of CDEM + Activin A (100 ng / ml). Incubate at 37

[1063] °C for a total of 48 h, replenishing medium after 24 h.

[1064] At this stage, assess the efficiency of definitive endoderm specification by performing flow cytometry using CXCR4 and CKIT.

[1065] Anterior foregut endoderm specification (Day 4 - 7)

[1066] 22. On day 3 of differentiation, prepare 1 Matrigel-coated 12-well plate and a half Matrigel-coated

[1067] 12-well plate (24 h prior to dissociating endoderm cells).

[1068] 23. Prepare CDEM + SB431542 (10 μM) + Dorsomorphin (2 μM) (DS / SB) + Y-27632 (10 μM) (for each 12-well plate, use 1 ml per well for plating).

[1069] 24. Take Matrigel-coated plate from the incubator and wash as in step 4 - 6.

[1070] 25. Aspirate medium from definitive endoderm day 4 cells and gently wash with 1 ml of

[1071] DMEM / F12 per well.

[1072] 26. Add 1 ml of Gentle Cell Dissociation Reagent and leave at room temperature for 2 - 3 min.

[1073] 27. Aspirate the Gentle Cell Dissociation Reagent with a P1000 and add 1 ml of DS / SB + CloneR2

[1074] (1:10) to each well. Carefully pipette up and down with a P1000 to detach cells from the plate surface but ensure colonies are preserved (4-5x) (do not pipette until a single cell suspension).

[1075] 28. Aspirate the DPBS from the Matrigel-coated 12-well plate and passage cells at a density of

[1076] 1:12-1:18. Note as the iPSC plating density is greater for endoderm specification, the monolayer on endoderm D4 contains many more cells. Use a ratio of 1:12, 1:15 or 1:18 to ensure efficient induction of NKX2.1 lung progenitors. Incubate at 37 °C for 24 h.

[1077] 29. After 24 h, aspirate the medium and add 1 ml per well of DS / SB medium without Clone R2.

[1078] Incubate at 37 °C for a total of 48 h, replenishing medium after 24 h. Note If using Y-27632 when splitting, expect more cell death than if using CloneR2 and adjust the splitting ratio accordingly. Test the different splitting ratios as mentioned above.

[1079] Induction of NKX2.1-expressing lung epithelial progenitors (Day 7 - 13)

[1080] 30. Aspirate the AFE induction medium and add 500 μl per well of CDEM + Wnt3a (100 ng / mL) +

[1081] R-Spondinl (250 ng / mL), BMP4 (10 ng / ml) + Retinoic Acid (100 nM).

[1082] 31. Replace medium every 24 h until day 13 of differentiation (Lung Day 6).

[1083] At this stage of differentiation, refer to the protocol for performing FACS on CPM+ lung epithelial progenitors.

[1084] Note CPM+ cells have shown to have a correlation with the confluent state of the wells. Depending on the confluency of the differentiation you may perform FACS on CPM+ lung epithelial progenitors starting on Lung Day 4, but the standard practice is for Lung Day 6.

[1085] Specification of lung progenitor cells to a distal lung fate (D15+)

[1086] For seeding of CPM+ cells into Matrigel droplets, ensure all steps are performed on ice to prevent quick solidifying of the Matrigel at room temperature. Ensure Matrigel is thawed on ice and coat the inside of the tips with cold PBS before handling Matrigel.

[1087] 32. Place a 48-well plate in the incubator to warm to 37°C.

[1088] 33. Determine the volume of cell suspension and Matrigel required using the calculations below:

[1089] Size of Matrigel droplet (50 μl) (1) x number of cells being seeded per pl of droplet (100- 400)

[1090] (2) = total number of cells required per droplet (3) Note we have been using 400 cells per ul of Matrigel droplet, which is 20,000 cells per droplet.

[1091] Number of cells collected (4) / total number of cells required per droplet (3) = total number of droplets that can be seeded (5)

[1092] • Total number of droplets that can be seeded (5) x size of Matrigel droplet (50 μl) (1) = total volume of Matrigel and cell suspension mix required (6)

[1093] 75% of total volume of Matrigel and cell suspension mix (6) = total volume of Matrigel required. Keep Matrigel frozen until needed.

[1094] 25% of total volume of Matrigel and cell suspension mix (6) = total volume of cell medium + Y-

[1095] 27632 (10 μM) required. Aliquot the desired volume of medium into a 1.5 ml Eppendorf tube and place in the fridge.

[1096] 34. Prepare CDEM + CHIR-99021 (3 μM) + FGF7 (10 ng / ml) + FGF10 (10 ng / ml) + cAMP (0.1 mM)

[1097] + IBMX (0.1 mM) + dexamethasone (50 nM) + Y-27632 (10 μM) + (CloneR2 (1:10). For a 48- well plate, prepare enough medium to add 300 μl per well. From this, also take an aliquot of medium required for resuspending cells and place in the fridge (see above).

[1098] 35. After performing FACS on CPM+ cells, centrifuge collected CPM(hi) cells for 5 min at 300 g.

[1099] 36. Take an aliquot of Matrigel and begin thawing on ice.

[1100] 37. Aspirate the supernatant, and gently resuspend the cell pellet in the aliquot of lung maturation medium prepared earlier, being careful not to introduce air bubbles.

[1101] 38. When the Matrigel has nearly thawed, take the desired volume with a coated pipette tip and carefully resuspend with the cell suspension, ensuring no air bubbles are introduced. Gently flick the tube to evenly disperse the cells within the matrix.

[1102] 39. Take the 48-well plate from the incubator and drop 50 μl of cell / matrix mix in the middle of the well.

[1103] 40. Place droplets at 37 °C for at least 10 min to allow the Matrigel to solidify.

[1104] 41. Add 300 μl per well of medium. Incubate at 37 °C for 24 h.

[1105] 42. After 48 h, replenish cells with distal lung maturation medium without CloneR2. Incubate at

[1106] 37 °C and refeed every 48 h.

[1107] Example 3 Differentiation of hPSCs to macrophages, DC, EC and FB

[1108] MATERIALS

[1109] Reagents

[1110] Dulbecco's Modified Eagle Medium (Corning, cat. no. 10-092-CM)

[1111] Recombinant human insulin (Sigma-Aldrich, cat. no. 11376497001)

[1112] Transferrin

[1113] Small molecule mix

[1114] Penicillin / Streptomycin

[1115] RPMI-1640 Medium (Sigma-Aldrich, cat. no. R8758)

[1116] B-27 supplement (50x; Thermo Fisher Scientific, cat. no. 17504044)

[1117] Non-essential Amino Acids

[1118] Iscove's Modified Dulbecco's Medium (IMDM; Thermo Fisher Scientific, cat. no. 12440053)

[1119] Ham's F12 medium (Corning, cat. no. 10-080-CV)

[1120] GlutaMAX (lOOx; Thermo Fisher Scientific, cat. no. 35050061)

[1121] 1-Thioglycerol (MTG; Sigma-Aldrich, cat. no. M6145)

[1122] • Ascorbic acid (Sigma-Aldrich, cat. no. A4544)

[1123] CHIR99021 Y-27632

[1124] Activin A

[1125] LY294002

[1126] SB431542

[1127] BMP4

[1128] VEGF

[1129] SCF

[1130] IL-3

[1131] IL-6

[1132] TPO

[1133] • TrypLE Express Enzyme (Thermo Fisher Scientific, cat. no. 12604021)

[1134] Dulbecco's Phosphate Buffer Solution (Thermo Fisher Scientific, cat. no. 14190144)

[1135] Nunc Tissue culture-treated T25 flasks (Thermo Fisher Scientific, cat. no. 156367)

[1136] Nunc Tissue culture-treated 12- and 48-well Multidishes (Thermo Fisher Scientific, cat. no.

[1137] 150628 and 150687)

[1138] Disposable sterile filter syringe (50 ml; BD Plastipak, cat. no. 300866)

[1139] Disposable Ministart sterile syringe filter (0.22 pl; Sartorius, cat. no,16532-K)

[1140] Snaplock Microcentrifuge tubes (1.5 ml; Axygen, cat. no. MCT-150-A)

[1141] Volume added

[1142] Medium Finalm Source (250ml final component concentration volume)

[1143] Iscove's modified Dulbecco's

[1144] IMDM medium (IMDM), no phenol red 117.25ml

[1145] (Gibco, cat no 21056-023) : Ham's F12 nutrient mix, GlutaMAX cally defined Ham's F12 supplement (Gibco, cat no 31765- 117.25ml sal medium 027)

[1146] Poly vinyl alcohol (Sigma-Aldrich,

[1147] PVA (5%) cat no P1836-250G) stock is 50ul lOug / ml

[1148] 5g / 100mL or 50mg / mL = 50ug / ul IL-6 Miltenyi, 130-093-931 50ng / ml

[1149] TPO Miltenyi, 130-095-747 50ng / ml dium: CDUM 100% m for IL-3 Miltenyi, 130-095-070 lOng / ml ytes IL-6 Miltenyi, 130-093-931 50ng / ml on M-CSF Miltenyi, 130-096-491 80ng / ml edium: m for CDUM 100% phage M-CSF Miltenyi, 130-096-491 80ng / ml on

[1150] CDUM 100% dium:

[1151] M-CSF (M0) Miltenyi, 130-096-491 80ng / ml m for

[1152] IFN-y (Ml) Peprotech, #300-02-100 20ng / ml phage

[1153] LPS (Ml) Sigma Aldrich, L5024 lOOng / ml ation

[1154] IL-4 (M2) Miltenyi, 130-093-922 20ng / ml

[1155] CDUM 100%

[1156] EGF Peprotech, AF100-15-1 lOng / ml dium:

[1157] Insulin Roche, 11376497001 5μg / ml m for

[1158] BMP4 Peprotech, #120-05-100 0.5nM ast induction

[1159] Isoprenaline Sigma Aldrich, 15627 2μg / ml

[1160] Hydrocortisone Sigma Aldrich, H2270 0.5μg / ml dium: CDUM 100% m for VEGF Peprotech, #100-20-500 300ng / mlhelial cell FGF2 Miltenyi, 130-093-842 200ng / ml on 8Bro-cAMP Sigma Aldrich, B7880 ImMedium:

[1161] CDUM 100% m for

[1162] GM-CSF Miltenyi, 130-093-865 50ng / ml ure dendritic

[1163] IL4 Miltenyi, 130-093-922 20ng / ml duction

[1164] CDUM 100% medium: TNF-a lOOng / ml

[1165] LPS Miltenyi, 130-094-023 lOOng / ml m for mature Sigma Aldrich, L5024 tic cells on iPSCs-derived macrophages (MΦ )

[1166] Passage hiPSCs when they reach 90% confluence as for the passaging for maintenance.

[1167] 1. Prepare a Matrigel-coated 12-well plate a day prior to seeding the cells for differentiation

[1168] 2. Prepare Homebrew (HB) E8 medium + Y-27632 (lOμM) (for a 12-well plate, use 1 ml per well for seeding)

[1169] 3. Take the plate out from the incubator, aspirate the DMEM / F12 and add 1 ml of DPBS into each well

[1170] 4. Use a P1000 to rinse each well by washing the DPBS across the surface 5 times, being careful not to scratch the Matrigel layer

[1171] 5. Return the 12-well plate with DPBS to the incubator to allow it to warm to 37°C

[1172] 6. Aspirate medium from the hPSCs culture, wash once with 5ml DPBS and add

[1173] 7. Dissociate hiPSCs using 2.5ml TryplE and incubate at 37°C, 4-5 minutes

[1174] 8. Check for the detachment of hPSCs from Matrigel-coated flask. At this stage cells should be floating, and colonies should be dispersed

[1175] 9. Add 3.5 ml DMEM to neutralise the TryplE and collect the cells by gently washing across the surface of the flask

[1176] 10. Transfer the cells to a 15ml falcon tube

[1177] 11. Take lOpI of cells suspension immediately and place it in a haemocytometer for cell counting

[1178] 12. Calculate the total number of cells required for seeding using calculation below

[1179] (Number of wells x surface area of well) x number of cells required per cm2= total number of cells required

[1180] 13. Take the number of cells required around 750 cell / cm2for seeding from step 10 and transfer to a 15ml falcon tube

[1181] 14. Centrifuge cells for 4 min at 300 g

[1182] 15. Aspirate the supernatant and gently resuspend the desired cells with HB E8 + Y-27632 medium

[1183] 16. Aspirate the DPBS from the 12-well plate and add 1 ml of cell suspension per well of the

[1184] Matrigel-coated 12-well plate

[1185] 17. Gently rock the plate from side to side and forward and backward to ensure even distribution of cells 18. Incubate the cells overnight at 37°C, 5% CO2

[1186] 19. Following 24 h from plating, remove medium and add 700pl of HB E8 without Y-27632 into each well

[1187] 20. Re-incubate the cells overnight at 37°C, 5% CO2

[1188] Mesoderm differentiation in 2 days

[1189] For all steps of mesoderm induction, use MD medium (reagent setup)

[1190] 21. Aspirate HB E8 medium and replace with 700 μl of MD medium

[1191] 22. Incubate the cells at 37°C, 5% CO2

[1192] Note: you might see many dead cells and only some colonies survive for the next day (many colonies survive will negatively affect the HPCs appearance, EC and FB generation)

[1193] 23. Aspirate the medium and add 700 μl per well of MD medium

[1194] 24. Incubate the cells at 37°C, 5% CO2

[1195] At this stage, assess the efficiency of mesoderm differentiation by performing flow cytometry using

[1196] CD140a.

[1197] Hemogenic endothelium induction in 3 days

[1198] For all subsequent steps of the differentiation protocol, use HE medium (refer to reagent set up)

[1199] 25. Aspirate medium from mesoderm differentiation day 2 and replace with 700 μl of HE medium

[1200] Note: Aspirate and refresh the medium gently since the cells are easily to detach

[1201] 26. Incubate the cells at 37°C, 5% CO2for a total of 72 h, leaving the medium until D5

[1202] At this stage, assess the efficiency of hemogenic endothelium induction by performing flow cytometry using CD144 and CD31.

[1203] Hematopoietic progenitor cells induction in 4 days

[1204] For all subsequent steps of the differentiation protocol, use HI medium (refer to reagent set up)

[1205] 27. Aspirate medium from hemogenic endothelium induction day 5 and replace with 700 μl of HI medium

[1206] 28. Incubate the cells at 37°C, 5% CO2

[1207] 29. Refresh medium after 48h

[1208] Note: pay attention on floating cells appearing in this stage

[1209] At this stage, assess the efficiency of blood progenitor cells induction by performing flow cytometry using CD43.

[1210] Monocyte induction in 6 days For all subsequent steps of the differentiation protocol, use Ml medium (refer to reagent set up)

[1211] 30. Collect all floating cells and transfer cell suspension into a 15-ml tube

[1212] 31. Wash each well with 1 ml Dulbecco's phosphate-buffered saline (DPBS) and collect it into the same 15-ml tube from step 30.

[1213] 32. Add 1 ml CDUM medium to each well. Pipet up and down three to four times to wash off the remaining cells and collect whole cells suspension in the same 15-ml tube from step 30

[1214] 33. Add 0.5ml TrypLE to each well and incubate at 37°C for 5 minutes. Tap several times and collect all cell suspension into the same 15-ml tube from step 30

[1215] 34. Add 0.5 ml prewarmed accutase to each well and incubate at at 37°C for 7 minutes

[1216] 35. Add 1 ml CDUM to each well then scrape off cells using a cell scraper and collect all suspension into the same 15-ml tube from step 30

[1217] Note: avoid pipetting too often and vigorously as it could damage the cells

[1218] 36. Add 1 ml CDUM to each well. Pipet up and down three to four times to wash off the remaining cells and collect all suspension into the same 15-ml tube from step 30

[1219] 37. Centrifuge cells collected in the 15-ml tube at 300 x g form 4 min at room temperature, then resuspend pellet in 12ml Ml medium

[1220] 38. Distribute lml cell suspension over twelve of a 24-well ultralow attachment plate

[1221] 39. Incubate the cell suspension 37°C, 5% CO2

[1222] 40. Refresh cells on D12 with Ml medium

[1223] Note: Aspirate only 50% medium and put another 1 ml Ml medium. Avoid disturbing the cells at the bottom.

[1224] 41. Incubate the cell suspension 37°C, 5% CO2

[1225] 42. Collect all the suspension cells and proceed to macrophages induction

[1226] At this stage, assess the efficiency of monocytes induction by performing flow cytometry using CD14.

[1227] Macrophages induction in 4 days

[1228] For all subsequent steps of the differentiation protocol, use M<t> medium (refer to reagent set up)

[1229] 43. On D14 of differentiation, prepare a FBS-coated 12-well plate and let stand overnight at 37 °C

[1230] 44. Collect all the suspension cells and transfer it into 15-ml tube

[1231] Note: pipet cell suspension up and down only twice using a 5-mi stripette gently. Too much resuspending will activate monocytes

[1232] 45. Filter the cell suspension through 100pm filter and centrifuge 300g for 3 minutes at room temperature

[1233] 46. Discard supernatant and Resuspend monocytes with 3ml CDUM medium 47. Count total cell number, remove FBS from 12-well plate and seed 40,000 cell / cm2for macrophages induction

[1234] Note: add 1 ml to each well and move plate in a cross-like pattern to distribute cells evenly. The rest of monocytes could be frozen using CryoStor CS10 cryopreservation medium to get a final concentration of 3.4 million / ml (aliquot 500pl into each cryovial)

[1235] 48. Incubate the cell at 37°C, 5% CO2

[1236] 49. Refresh cell on D17 with M<t> medium

[1237] Note: do not disturb the cells during the first two days. Only ~10% cells adhere after 2 days which is normal. The cells should be confluent after 4 - 6 days with elongated morphology. Refresh only 50% medium and add 1 ml Md> medium

[1238] Polarization can be done if the cells reach > 80% confluencyfor M0 macrophages

[1239] At this stage, assess the efficiency of M<t> induction by performing flow cytometry using CDllb.

[1240] Polarization induction in 2 days

[1241] For all subsequent steps of the differentiation protocol, use Pol medium (refer to reagent set up)

[1242] 50. Aspirate the medium from the plate and refresh the cells with pol medium for MO, Ml and

[1243] M2

[1244] 51. Incubate the cell at 37°C, 5% CO2for 48h

[1245] Note: upon polarization the cell morphology changes into stellar shape for Ml and rounded cells for

[1246] M2

[1247] At this stage, assess the efficiency of M<t> polarization by performing flow cytometry using CDllb, CD80 and CD86. iPSCs-derived dendritic cells (DC)

[1248] Immature dendritic cells induction in 6 days

[1249] For all subsequent steps of the differentiation protocol, use iDC medium (refer to reagent set up), do the same differentiation protocol to generate monocytes

[1250] 1. On D14 of differentiation, prepare a FBS-coated 12-well plate and let stand overnight at 37 °C

[1251] 2. Collect all the suspension cells and transfer it into 15-ml tube

[1252] Note: pipet cell suspension up and down only twice using a 5-ml stripette gently. Too much resuspending will activate monocytes

[1253] 3. Filter the cell suspension through 100pm filter and centrifuge 300g for 3 minutes at room temperature 4. Discard supernatant and Resuspend monocytes with 3ml CDUM medium

[1254] 5. Count total cell number, remove FBS from 12-well plate and seed 40,000 cell / cm2for immature dendritic cells induction

[1255] Note: add 1 ml to each well and move plate in a cross-like pattern to distribute cells evenly

[1256] 6. Incubate the cell at 37°C, 5% CO2for 72h

[1257] 7. Refresh cell with iDC medium

[1258] Note: transfer all the suspension cells in each well into 15-ml tube and centrifuge 300g for 3 minutes, aspirate the supernatant and resuspend the cells gently with fresh IDC medium and distribute the cells back to the plate (1 ml per well)

[1259] 8. Incubate the cell at 37°C, 5% CO2

[1260] At this stage, assess the efficiency of iDC induction by performing flow cytometry using CDllc, CD86 and MHC-II / HLA-DR.

[1261] Mature dendritic cells induction in 2 - 6 days

[1262] For all subsequent steps of the differentiation protocol, use mDC medium (refer to reagent set up)

[1263] 9. Refresh cell with mDC medium

[1264] Note: transfer all the suspension cells in each well into 15-ml tube and centrifuge 300g for 3 minutes, aspirate the supernatant and resuspend the cells gently with fresh mDC medium and distribute the cells back to the plate (1 ml per well)

[1265] 10. Incubate the cell at 37°C, 5% CO2

[1266] Note: Incubation time might vary depending on purposes, 2 days is enough for analysis and 6 day is to see obvious DC morphology with dendrites

[1267] At this stage, assess the efficiency of iDC induction by performing flow cytometry using CDllc, CD86 and MHC-II / HLA-DR. iPSCs-derived endothelial cells (EC)

[1268] Endothelial cells induction in 7 days

[1269] For all subsequent steps of the differentiation protocol, use EC medium (refer to reagent set up), do the same differentiation protocol up to mesoderm induction

[1270] 1. Aspirate medium from mesoderm differentiation day 2 and replace with 700 μl of EC medium

[1271] 2. Incubate the cells at 37°C, 5% CO2for a total of 72 h, leaving the medium until D5

[1272] 3. Aspirate medium and refresh the cells with EC medium 4. Incubate the cells at 37°C, 5% CO2up to D9

[1273] At this stage, assess the efficiency of EC induction by performing flow cytometry using CD144 and

[1274] CD31. iPSCs-derived fibroblast (FB)

[1275] Fibroblast induction in 9 days

[1276] For all subsequent steps of the differentiation protocol, use FB medium (refer to reagent set up), do the same differentiation protocol up to mesoderm induction

[1277] 1. Aspirate medium from mesoderm differentiation day 2 and replace with 700 μl of FB medium

[1278] 2. Incubate the cells at 37°C, 5% CO2

[1279] 3. Aspirate medium and refresh the cells with FB medium every other day until Dll

[1280] 4. Incubate the cells at 37°C, 5% CO2

[1281] At this stage, assess the efficiency of FB induction by performing flow cytometry using CD44, CD73,

[1282] CD90, CD140a, CD13, and FB / FSP1.

[1283] Example 3 - Differentiation of hiPSCs to T cells from hematopoietic progenitor cells

[1284] We co-cultured HPCs with stromal cells 0P9-DL1 cells in the presence of SCF, IL-7, TPO, FLT3L for 14 days to promote T cells generation (Fig. 17a). HPCs showed morphological transition from small- rounded cells to floating large-rounded cells at the end point of differentiation (D27) (Fig. 17b). hiPSCs- derived HPCs developed to pro T cell stage by expressing CD45, CD7 after co-culturing with OP9-DL1 cells for 12 days. We observed that ~30% of T cells expressed double positive for CD45 and CD7 and approximately 95% of them was single positive for CD45 (Fig. 17c). Hence, these findings showed that the HPCs generated by using our protocol were definitive blood progenitor cells, distinguishing them from primitive HPCs. However, T cells expressing CD45+CD7+needed further stimulation to generate

[1285] CD4CD8 double positive (DP) T cells followed by generation of mature CDS single positive (SP) T cells.

[1286] Directed differentiation of hiPSCs into T cells

[1287] After generating HPCs according to the invention, for T cells differentiation, 52,000 cells / cm2of HPCs were cocultured with stromal cells OP9-DL1, which had been plated 2 days before co-culture at density

[1288] 5,000 cells / cm2in 6-well plate, in a-MEM medium (Table 6) supplemented with 10% FBS, Mono-thio glycerol (100 μM), SCF (10 ng / ml), IL-7 (25ng / ml), TPO (50 ng / ml), and FLT3L (10 ng / ml) for 12 days.

[1289] The medium was replaced every 3 days up to D14. At this stage, the T cell differentiation efficiency was assessed for CD45 and CD7 expression using flow cytometry.

[1290] Example 4 - Three-dimensional (3D) co-culture of M<D, DCs, FLCs, VECs, and HLCs for modelling hiPSCs-derived liver tissue

[1291] Introduction - 3D co-culture technology for modelling hiPSCs-derived liver tissue

[1292] Human induced pluripotent stem cells (hiPSCs) provide a platform that can be used for disease modelling and developing a better understanding of developmental biology. The traditional two- dimensional (2D) co-culture of different cell types derived from hiPSCs is the simplest platform for modelling multicellular tissue similar to an in vitro model, even though two dimensional multilineage co-culture presents a more accurate model than monoculture to mimic tissue of interest. For example.

[1293] 2D co-culture of astrocytes and neurons derived from hiPSCs recapitulates the brain neuronal-cell network and hiPSC-derived glial-neuronal co-culture enhances cellular maturity and potential propagation (Wen et al., 2014; Ishii et al., 2017). There are limitations to 2D co-culture technology; the most obvious of these is loss of the tissue complexity where the cells normally grow in 3D environment in vivo especially the parenchymal cells colonize and proliferate the living organs or tissues in highly complex 3D environment supported by ECM and other cell types (Gattazzo et al., 2014). A lack of 3D environmental cues showed also the immaturity 2D culture derivatives and displayed foetal tissues characteristics than adult in many cases (S. M. Wu & Hochedlinger, 2011). Therefore, there is a clear need for more complex 3D model systems to recapitulate and mimic physiological in vivo system.

[1294] One of the 3D systems, apart from 3D bioengineered tissue using scaffold and organ on chip technology, is organoids which are miniaturized 3D and self-organizing cellular aggregates sometimes consisting of multiple cell types to represent structurally the analogue of human organs and tissues.

[1295] Hence, 3D organoids provide physiologically relevant models for developmental biology and diseases when compared to traditional 2D co-culture (Takebe & Wells, 2019).

[1296] As the largest solid organ in the human body, the liver plays important roles not only for the digestive system but also regulating the whole-body energy metabolism. Given its function in the circulatory system, the liver receives all materials and compounds from the intestine, allowing the liver to be the place for detoxification and for immune response to kill invading microbes. Hence, the liver is also exposed with antigens from systemic circulation through liver arterial vessel (Kzhyshkowska et al., 2012). Since the functions and structures of the liver are heterogenous, the composition of cell types comprising the liver is also diverse. Hepatocytes, the parenchymal cells, are most of the cells living in the liver, comprising around 60% of the organ's total cells and 80% of its volume. Non-parenchymal cells (NPCs) are around 40% of liver cells, consisting of 20% of sinusoidal endothelial cells (LSECs), 15% of Kupffer cells, and 5-8% hepatic stellate cells (HSCs). While other immune cell populations are only minor fraction of NPCs such as natural killer T (NKT) cells (Malarkey et al., 2005; Seo & Jeong, 2016).

[1297] The majority of liver functions is performed by hepatocytes, however liver macrophages also play important roles in maintaining organ homeostasis (Duarte et al., 2015; Krenkel & Tacke, 2017).

[1298] Therefore, it is required for both liver parenchymal and non-parenchymal cells to recapitulate liver tissue in vitro, which is either obtained from primary liver tissue or generated from hiPSCs.

[1299] Generation of three-dimensional (3D) multi-tissue liver organoids (mLOs) derived from hiPSCs offers an alternative model to study liver development and disease (Jin et al., 2021; Telles-Silva et al., 2022).

[1300] A recent study showed that mLOs consisting of parenchymal (HLCs) and non-parenchymal cells (HSCs,

[1301] ECs) displayed functional microvasculature of mLOs by modulating Notch signalling, allowing the precise modelling of liver tissue in vitro (H. J. Kim et al., 2023). However, this study still missed one important cell type which was Kupffer cells to reflect complex interactions between diverse liver cell types to model physiologically and pathologically relevant liver tissue.

[1302] Our focus for this Example 4 was to present an example of how the cells generated in the previous

[1303] Examples 1-2 can be combined to make a multi-tissue, immune competent organoid. The exemplar tissue was the liver and we generated multi-tissue liver organoids (mLOs) by integrating both parenchymal (HLCs) and NPCs (M<t>, DCs, VECs, and FLCs) at the ratios that are consistent with normal cellular composition of the liver to physiologically model liver tissue using xeno-free and chemically defined medium. Therefore, the aim of this Example 4 is to:

[1304] 1. Generate hepatocytes like cells (HLCs) from hiPSCs

[1305] 2. Generate mLOs by integrating different cell types

[1306] We hypothesised that mLOs could be generated using xeno-free and chemically defined medium that will ultimately be physiologically relevant to liver tissue in in vivo system.

[1307] Methods

[1308] Generation of multilineage liver organoids (mLOs). After generating all cells of interest from hiPSCs according to the invention herein (cf. other Examples 1-3), all cells were put together in pyramid-shape microwells plate called aggrewell (Stemcell Technologies, #34815) with 900,000 cells in total according to manufacturer's instruction and cultured in CDEM for the first 5 days to let the organoid selfassembly in the presence of oncostatin M (30 ng / ml), HGF (50 ngm / ml), M-CSF (80 ng / ml) and cloneR2

[1309] (Stemcell Technologies, #100-0691) (10% v / v). The organoids were then transferred onto 24-well ultralow attachment plate using with splitting ratio 1:2 for 11 days supplemented only with oncostatin M

[1310] (30 ng / ml) (Fig. 2-6). The human normal liver cells ratio was used for co-culture as follows hepatocytes

[1311] : endothelial cells : Kupffer cells : hepatic stellate cells : dendritic cells (12 : 4 : 3 : 1 : 1). Next, the organoids were harvested for the next assay. Furthermore, for fatty liver disease model, we treated

[1312] HLCs with 300 μM of palmitic acid (PA) for 48 h before combined with NPCs.

[1313] Results

[1314] Differentiation of hiPSCs into hepatocytes like cells (HLCs) in chemically defined and xeno-free medium (CDEM)

[1315] After successfully establishing a platform to differentiate hiPSCs into M<D, DC, FLCs, and VECs using chemically defined and xeno-free medium, we decided to also generate hepatocytes like cells (HLCs) using our chemically defined and xeno-free medium according to the invention herein (CDEM) (Fig. lOf). To do this, we seeded hiPSCs on Matrigel™ coated dishes using optimized seeding density of

[1316] 20,000 cells / cm2in CDEM for endoderm cells origin. To differentiate hiPSCs into definitive endoderm

[1317] (DE) population, we used a combination of Activin A, CHIR99021, and LY294002 to enhance the Wnt signalling and inhibit the PI3K pathway in the cells for 4 days. In this stage, the cells morphology was completely monolayer of petal / cobblestoned-shape cells and confluent in whole surface area of the dish. The next stage is anterior foregut (AF) induction stimulated by only the addition of Activin A for

[1318] 2 days in the medium. There was obvious morphological transition from DE to AF where the cells had flattened, looked rounder and resembled the early phase of HLCs in which nuclei became more prominent. Furthermore, the AF population was then stimulated into hepatic endoderm cells using

[1319] SB431542, BMP4, and FGF10 for 4 days and the cells became more compact with few formations of three-dimensional cell cluster. The next stage is maturation step for 14 days where the morphology of the cells completely became polygonal, multinucleated HLCs with lipid droplet production (Fig. 10a- b).

[1320] To assess the capacity of hiPSCs differentiation into definitive endoderm (DE) cells, we used CXCR4 and cKIT co-expression to quantify DE efficiency using flow cytometry analysis. It showed that the combination of small molecules and growth factor used in this stage was efficient to enhance DE induction, leading to a mean percentage of 99.64% CXCR4+cKIT+cells (n=3) (Fig. 10g). By the end at

[1321] D24, we also assessed generated HLCs by flow cytometry using mature marker of hepatocytes ASGPR1 co-stained with EpCAM. The analysis showed that almost 100% of HLCs was EpCAM+cells (epithelial marker) and around 70% of the HLCs was double EpCAM+ASGPRl+cells (Fig. 5-2b). Furthermore, we also checked the genes expression profile of HLCs at D24 using qPCR analysis. We found that liver maturation markers such as ALB, AFP, A1AT, HNF4A, CYP3A7 was upregulated during maturation step compared to undifferentiated hiPSCs BE31 (Fig. lOe, lOh).

[1322] Generation of multi-tissue liver organoids (mLOs) in chemically defined and xeno-free medium

[1323] (CDEM)

[1324] To generate multi-tissue liver organoids (mLOs) consisting of different cell types, we first integrated all targeted cells from mesoderm origin (M<t>, DC, FLCs, and VECs) and endoderm origin (HLCs) in pyramidshape microwells with ratio of five different cell types based on the percentage of normal human liver cell composition (HLCs / VECs / M<t> / DC / FLCs, 12 : 4 : 3 : 1 : 1) and incubated the co-cultured cells in the presence of HGF, One M, and M-CSF using CDEM to let the cells self-aggregate for 5 days and further transferred into ultra-low attachment (ULA) plates to let self-assemble of mLOs supplemented only with One M for 11 days (Fig. lOi). After 5 days of self-aggregation, the mLOs became compact with some loose cells at the edges of the organoids. Whereas mLOs size increased gradually after transferring into ULA plate, the mLOs grew as individual organoids in D8 and they merged and formed huge colonies by the end of D16 (Fig. 10L). To ensure all the cell types were still alive after integration. we then dissociated mLOs into single cells and stained them with each cell specific markers in which

[1325] HLCs were detected by the expression of EpCAM, VECs with CD31 expression, M<5 with CDllb expression, FLCs with FSP1 expression, and DCs with HLADR expression (Fig. 10m). According to flow cytometry analysis, the percentage of each cell types (HLCs, VECs, M<D, FLCs, and DCs) was still within the ratio of normal human liver tissue. There was around 60% EpCAM+cells, 25% CD31+cells, 20%

[1326] CDllb+cells, 4% FSP1+cells, and 10% HLADR+cells (n=2) (Fig. lOn).

[1327] Single-cell transcriptome analysis of hiPSC-derived mLOs

[1328] To further understand the cellular diversity within the mLOs, we performed single-cell transcriptomic signatures by scRNAseq. A total of 14 clusters were detected and systematically assigned to hepatic cell types by unique markers. Normal mLOs were used as control and then compared to mLOs treated with palmitic acid (PA) as fatty liver disease model (Fig. 5-5a). Furthermore, the dot plot showed the genes list associated with hepatic cells where cluster 2 was mainly indicated as hepatocyte-like population with high expression of AFP, SERPINA1, HNF4A, KRT18, and PROXI. Cluster 13 was indicated as hepatic-like immune cells with enrichment of PTPRC (CD45), CD68, CCL3, CD36, and

[1329] PECAM1 whereas cluster 14 was indicated as hepatic-like fibroblast cells which upregulated ICAM1 and CD36. In addition, the upregulation of genes related to cholangiocytes especially SLC4A5 was in cluster 4, 5, 9, 10, and 11 (Fig. 5-5b). Furthermore, PA-treated mLOs in cluster 13 indicated as hepatic- like immune cells highly expressed ICAM1, CD36, and PECAM1 whereas PA-treated mLOS in cluster 14 indicated as hepatic-like fibroblast was enriched with SLC4A5, KRT8, ICAM1, and CD36 compared to control (normal mLOs) (Fig. 5-5c).

[1330] To specify the hepatic population within control mLOs and PA-treated mLOs, we then did manual annotation to cluster the population. There were four main populations identified: cholangiocytes. hepatocytes, Kupffer cells, and stellate cells (Fig. 5-6a). Hepatocytes highly expressed AFP, SERPINA1,

[1331] HNF4A, KRT18, and PROXI. Additionally, some expression of cholangiocyte-related genes (KRT19 and

[1332] SLC4A5') and stellate cells-related genes (ICAM1 and KRT8) was also significantly upregulated in hepatocytes populations. Kupffer cell predominantly expressed three (PTPRC / CD45, CD68, and CCL3) and two specific genes 1.CD36, PECAM1) for EC and one specific gene (CD86) for DC, while cholangiocytes upregulated SLC4A5. ICAM1 and CD36 was strongly expressed in stellate cells (Fig. 5-

[1333] 6b). The comparison between PA-treated mLOs and control showed distinct expression of hepatic genes especially in Kupffer cell population. In PA-treated mLOs, Kupffer cells upregulated CD36,

[1334] PECAM1, and CCL3 compared to control (Fig. 5-6c). Furthermore, each annotated cluster suggested the enrichment of top 10 specific markers for cholangiocytes and stellate cells, whereas Kupffer cells and hepatocytes predominantly expressed not only their markers but also cholangiocytes markers (Fig.

[1335] 5-6d).

[1336] To determine whether the normal mLOs acquired a transcriptional signature resembling human liver tissue, we compared them with online healthy liver dataset (Guilliams et al., 2022) using celltypist annotation (Xu et aL, 2023) (Fig. 5-7a). It showed that Kupffer cells from manual annotation was significantly like macrophages from celltypist annotation whereas cholangiocytes in manual annotation was also similar to cholangiocytes in celltypist annotation.

[1337] Discussion

[1338] Here, our findings provide an optimized method to generate HLCs from hiPSCs in fully defined and xeno-free medium using a protocol with high reproducibility. These hiPSC-derived HLCs were then combined with cells generated from mesoderm origin (M<t>, DCs, FLCs, and VECs) to generate multilineage liver organoids (mLOs). In fully defined and xeno-free medium, we observed efficient definitive endoderm (DE) induction using a combination of CHIR99021, LY294002, and Activin A. This was in line with our findings that DE induction could be maximized using one-day treatment with CHIR99021, transient PI3K inhibition, and continuous activin A signalling (Naujok et aL, 2014; Teo et al., 2014). From the optimization step, we found that 3 μM was sufficient to induce DE population without inducing any toxicity to the cells grown in serum-free medium. Previous study showed that

[1339] CHIR improved the differentiation efficiency in the absence of serum and albumin, but higher dose can induce cell toxicity (Lian et aL, 2015). Furthermore, flow cytometry analysis revealed that the DE population was enriched with the upregulation of CXCR4 and cKIT expression. This was relevant to previous studies suggesting that hPSCs treated with activin A and Wnt3a for 4 days highly expressed

[1340] CXCR4, CD117 (cKIT), and EpCAM and were lack of contaminating mesodermal cells (Wong et al., 2010;

[1341] Holtzinger et al., 2015). After DE stage, the cells were then induced to anterior foregut formation using

[1342] 50 ng / ml of activin A only for 48 h, which was sufficient to generate FG population with petal-shape morphology, which was relevant to a study showed that the expression of foregut markers such as

[1343] HNF4A, HHEX, 50X17, and GATA4 could be induced by using 50 ng / ml of activin A only which was comparable to 200 ng / ml of activin A (Hannan et al., 2013). The FG population was then proceeded to hepatic endoderm induction in the presence of SB431542, BMP4, and FGFlOfor 4 days. It was reported that hepatic endoderm (HE) could be derived from DE population by the combination of BMP4 and

[1344] SB431542 for 4 days, which highly expressed CD13 and EpCAM (Kim et aL, 2022). BMP signalling was essential for early stage of hepatic induction which regulated hepato-pancreatobilliary boundary patterning by indirectly repressing SOX9 expression in HE (Gordillo et aL, 2015; Palaria et al., 2019).

[1345] Furthermore, to generate HLCs from HE, two growth factors were routinely added to the medium: hepatocyte growth factor (HGF) and oncostatin M (One M), these two GFs can affect proliferation, phenotype, and metabolism of hepatocytes (Hay et aL, 2008; Si-Tayeb et aL, 2010; Hannan et aL,

[1346] 2013). Previous studies suggested that HGF can initiate the differentiation of hiPSCs into hepatoblasts whereas One M can inhibit their proliferation while promoting the maturation instead via signal transducer and activator of transcription 3 (STAT3) signaling pathway and these two GFs could direct the differentiation into hepatic lineage while inhibiting the development of bile duct (Hu et aL, 1993;

[1347] Kamiya et al., 2001; Suzuki et al., 2003).

[1348] Furthermore, hiPSC-derived HLCs generated using in-house optimized differentiation protocol in fully chemically and xeno-free medium were characterized by flow cytometry using hepatocyte surface marker asialoglycoprotein receptor 1 {ASGPR1 / ASGRT) and the expression of hepatocytes-specific genes. ASGPR1 is expressed in adult liver tissue and extremely low in foetal liver, and is highly expressed in maturation stage of HLCs differentiation (Takayama et aL, 2014; Peters et aL, 2016).

[1349] Previous studies showed that ASGPR1 was used to enrich HLCs since the differentiation efficiency only ranged from 4.2% to 48% of ASGPR1+cells (Basma et al., 2009; Peters et al., 2016). While using our optimized HLCs-directed differentiation protocol in in-house medium, we could generate HLCs with

[1350] 70% ASGPR1+cells, indicating that the protocol and medium used worked efficiently without any enrichment step. Next, we analysed the expression of hepatocyte-specific markers in HLCs D24 such as albumin (ALB), alpha fetoprotein (AFP), alpha-1 antitrypsin (A1AT / SERPINA1), hepatocytes nuclear factor 4 alpha (HNF4a), and cytochrome P450 family 3 subfamily A member 7 (£YP3A?Y HLCs derived from hiPSCs typically still expressed foetal expression (AFP) compared to adult hepatocytes but it will significantly drop during maturation step when they show functional capabilities including ALB secretion, urea secretion, and CYP3A4 activity representing the function of liver in secretory and detoxification.

[1351] Based on the previous studies hiPSC-derived mLOs can be produced by integration of parenchymal

[1352] (hepatic endoderm / hepatoblasts (HE)) and non-parenchymal cells such as mesenchymal stem cells

[1353] (MSCs), hepatic stellate cells (HscLCs), endothelial cells (ECs), supporting parenchymal cells maturation and function through paracrine communication and direct cell-cell interaction (Tsang et al., 2021; Kim et al., 2023). However, the limitation of those studies was the absence of immune cells such as M<t> and DCs in mLOs. Therefore, we generated mLOs which composed of parenchymal cell (HLCs) and nonparenchymal cells (VECs and FLCs) including immune cells (M<t> and DCs) in normal ratio of human liver cells, which can make the hepatic niche more complex and accurate to model normal human liver.

[1354] Based on single-cell RNA sequencing, we found 14 clusters in the mLOs where only 3 clusters that could be identified based on upregulation of their specific markers even though some markers was also found in the other cluster of cells: AFP, SERPINA1, HNF4A, KRT18, and PROXI (hepatocyte-like population: cluster 2) (Wesley et al., 2022); PTPRC, CD68, CCL3, CD36, PECAM1 (hepatic-like immune cells: cluster 13) (MacParland et al., 2018); ICAM1 and CD36 (hepatic-like fibroblasts: cluster 14)

[1355] (Hellerbrand et al., 1996; Schneiderhan et al., 2001). To further analyse the data, we compared the gene expression from manual annotated normal mLOs as control to PA-treated mLOs and found higher expression of pro-inflammatory marker CCL3 in PA-treated mLOs than control, which was in line with previous studies suggested that macrophages inflammatory protein-1 alpha (MIP-la / CCL3) secreted by M<D was responsible for pathogenesis various inflammatory diseases such as rheumatoid arthritis, multiple myeloma, NAFLD, etc (Dapunt et al., 2014; Preedy & Patel, 2015; Nagata et al., 2022).

[1356] Example 5 - Cardiac differentiation Cells were cultured in standard cell culture conditions (5% CO2, 37 °C, humidified ambient). 12,000 cells / cm2C3G3 or MoPCK hPSCs were cultured in E8 medium for 2 days. They were then cultured in

[1357] CDUM supplemented with 1 ng / mL BMP4 and 1% (v / v) Matrigel™ After 1 day, the medium was changed to CDUM supplemented with 8 ng / mL Activin A and 10 ng / mL BMP4. After two further days, the medium was changed to CDUM supplemented with 10 μM KY0211 and 10 μM XAV939. After two further days, the medium was changed to fresh CDUM supplemented with 10 μM KY0211 and 10 μM

[1358] XAV939. After two further days, the medium was changed to CDUM. The cells were kept in culture for a further 15 days, refreshing the medium.

[1359] Medium Index

[1360] The Phenol Red-free IMDM may comprise or consist of the components of Table 1:

[1361] Component Mol. weight Cone. (mg / L) Cone. (mM)

[1362] Glycine 75 30 0.4

[1363] L-Alanine 89 25 0.28089887

[1364] L-Arginine hydrochloride 211 84 0.39810428

[1365] L-Asparagine (freebase) 150 25 0.16666667

[1366] L-Aspartic acid 133 30 0.22556391

[1367] L-Cystine 2HCI 240 91.4 0.38083333

[1368] L-Glutamic Acid 147 75 0.5102041

[1369] L-Glutamine 146 584 4

[1370] L-Histidine hydrochloride-H2O 210 42 0.2

[1371] L-lsoleucine 131 105 0.8015267

[1372] L-Leucine 131 105 0.8015267

[1373] L-Lysine hydrochloride 183 146 0.7978142

[1374] L-Methionine 149 30 0.20134228

[1375] L-Phenylalanine 165 66 0.4

[1376] L-Proline 115 40 0.3478261

[1377] L-Serine 105 42 0.4

[1378] L-Threonine 119 95 0.79831934

[1379] L-Tryptophan 204 16 0.078431375

[1380] L-Tyrosine disodium salt 225 104 0.46222222

[1381] L-Valine 117 94 0.8034188

[1382] Biotin 244 0.013 5.33E-05

[1383] Choline chloride 140 4 0.028571429

[1384] D-Calcium pantothenate 477 4 0.008385744

[1385] Folic Acid 441 4 0.009070295

[1386] Niacinamide 122 4 0.032786883

[1387] Pyridoxal hydrochloride 204 4 0.019607844

[1388] Riboflavin 376 0.4 0.00106383

[1389] Thiamine hydrochloride 337 4 0.011869436 Vitamin B12 1355 0.013 9.59E-06 i-lnositol 180 7.2 0.04

[1390] Calcium Chloride (CaCI2) (anhyd.) 111 165 1.4864864

[1391] Magnesium Sulfate (MgSO4) (anhyd.) 120 97.67 0.8139166

[1392] Potassium Chloride (KCI) 75 330 4.4

[1393] Potassium Nitrate (KNOg) 101 0.076 7.52E-04

[1394] Sodium Bicarbonate (NaHCOg) 84 3024 36

[1395] Sodium Chloride (NaCI) 58 4500 77.586205

[1396] Sodium Phosphate monobasic 138 125 0.9057971

[1397] (NaH2PO4-H2O)

[1398] Sodium Selenite (Na2SeO3-5H20) 263 0.0173 6.58E-05

[1399] D-Glucose (Dextrose) 180 4500 25

[1400] HEPES 238 5958 25.033613

[1401] Sodium Pyruvate 110 110 1

[1402] Table 1.

[1403] The IMDM that comprises Phenol Red may comprise or consist of the components of Table 2:

[1404] Component Mol. weight Cone. (mg / L) Cone. (mM)

[1405] Glycine 75 30 0.4

[1406] L-Alanine 89 25 0.28089887

[1407] L-Arginine hydrochloride 211 84 0.39810428

[1408] L-Asparagine (freebase) 132 25 0.18939394

[1409] L-Aspartic acid 133 30 0.22556391

[1410] L-Cystine-2HCI 313 91.4 0.29201278

[1411] L-Glutamic Acid 147 75 0.5102041

[1412] L-Glutamine 146 584 4

[1413] L-Histidine hydrochloride-H2O 210 42 0.2

[1414] L-lsoleucine 131 105 0.8015267

[1415] L-Leucine 131 105 0.8015267

[1416] L-Lysine hydrochloride 183 146 0.7978142

[1417] L-Methionine 149 30 0.20134228

[1418] L-Phenylalanine 165 66 0.4 L-Proline 115 40 0.3478261

[1419] L-Serine 105 42 0.4

[1420] L-Threonine 119 95 0.79831934

[1421] L-Tryptophan 204 16 0.078431375

[1422] L-Tyrosine disodium salt 225 104 0.46222222

[1423] L-Valine 117 94 0.8034188

[1424] Biotin 244 0.013 5.33E-05

[1425] Choline chloride 140 4 0.028571429

[1426] D-Calcium pantothenate 477 4 0.008385744

[1427] Folic Acid 441 4 0.009070295

[1428] Niacinamide 122 4 0.032786883

[1429] Pyridoxal hydrochloride 204 4 0.019607844

[1430] Riboflavin 376 0.4 0.00106383

[1431] Thiamine hydrochloride 337 4 0.011869436

[1432] Vitamin B12 1355 0.013 9.59E-06 i-lnositol 180 7.2 0.04

[1433] Calcium Chloride (CaCI2) (anhyd.) 111 165 1.4864864

[1434] Magnesium Sulfate (MgSO4) (anhyd.) 120 97.67 0.8139166

[1435] Potassium Chloride (KCI) 75 330 4.4

[1436] Potassium Nitrate (KNO3) 101 0.076 7.52E-04

[1437] Sodium Bicarbonate (NaHCO3) 84 3024 36

[1438] Sodium Chloride (NaCI) 58 4505 77.67242

[1439] Sodium Phosphate monobasic 138 125 0.9057971

[1440] (NaH2PO4-H2O)

[1441] Sodium Selenite (Na25eO3-5H20) 173 0.017 9.83E-05

[1442] D-Glucose (Dextrose) 180 4500 25

[1443] HEPES 238 5958 25.033613

[1444] Phenol Red 376.4 15 0.039851222

[1445] Sodium Pyruvate 110 110 1

[1446] Table 2.

[1447] The L-alanyl-L-glutamine-free F12 may comprise or consist of the components of Table 3: Folic Acid 1.3 i-lnositol 18.02

[1448] Nicotinamide 0.037

[1449] Pyridoxine-HCI 0.062

[1450] Riboflavin 0.038

[1451] Thiamine-HCI 0.34

[1452] Vitamin B12 1.36

[1453] Dextrose 1802

[1454] Hypoxanthine, Sodium 4.77

[1455] DL-Thioctic (lipoic) acid 0.21

[1456] Methyl Lineoleate 0.088

[1457] Phenol Red, Sodium 1.2

[1458] Putrescine-2HCI 0.16

[1459] Sodium Pyruvate 110

[1460] Thymidine 0.73

[1461] Table 3.

[1462] The F12 comprising L-alanyl-L-glutamine may comprise or consist of the components of Table 4:

[1463] Component Cone. (mg / L)

[1464] Glycine 7.5

[1465] L-Alanine 8.9

[1466] L-Alanyl-L-Glutamine 217

[1467] L-Arginine hydrochloride 211

[1468] L-Asparagine H2O 15.01

[1469] L-Aspartic acid 13.3

[1470] L-Cysteine hydrochloride-H2O 35.12

[1471] L-Glutamic Acid 14.7

[1472] L-Histidine hydrochloride-H2O 21

[1473] L-lsoleucine 4

[1474] L-Leucine 13.1

[1475] L-Lysine hydrochloride 36.5

[1476] L-Methionine 4.5 Thymidine 0.7

[1477] Table 4.

[1478] Components Molecular Concentration mM

[1479] Weight (mg / L)

[1480] Arachidonic Acid 304.74 2 0.006563

[1481] Cholesterol 386.65 220 0.56899

[1482] DL-alpha-Tocopherol 472.74 70 0.148073

[1483] Acetate

[1484] Linoleic Acid 280.45 10 0.035657

[1485] Linolenic Acid 278.44 10 0.035914

[1486] Myristic Acid 228.38 10 0.043787

[1487] Oleic Acid 282.47 10 0.035402

[1488] Palmitic Acid 256.43 10 0.038997

[1489] Palmitoleic Acid 254.41 10 0.039307

[1490] Pluronic F-68 7680 90000 11.71875

[1491] Stearic Acid 284.48 10 0.035152

[1492] Tween 80® 1310 2200 1.679389

[1493] Table 5. Chemically defined lipid concentrate

[1494] The MEM a may ...

Claims

CLAIMS1. A chemically defined universal medium (CDUM) comprising one or more of: a) a first basal medium, such as Iscove's Modified Dulbecco's Medium (IMDM); b) a second basal medium comprising a nutrient mixture comprising amino acids, vitamins and inorganic salts; c) a polymer, such as poly(vinyl alcohol) (PVA); d) lipids; e) an activator of the ERK / MAPK pathway, such as insulin; f) an iron carrier; g) selenium supplement; h) a cell growth and proliferation promoter, such as ethanolamine; i) a first anti-oxidant, such as monothioglycerol; j) a second anti-oxidant, such as ascorbic acid-2-phosphate (AA2P); k) L-glutamine supplement.

2. The chemically defined universal medium according to claim 1, wherein the chemically defined medium (CDUM) does not comprise albumin.

3. The chemically defined universal medium according to claim 1 or claim 2, further comprising a nonessential amino acids supplement.

4. The chemically defined universal medium according to any preceding claim, further comprising at least one antibiotic.

5. The chemically defined universal medium according to any preceding claim, wherein the basal medium comprises or consists of Iscove's Modified Dulbecco's Medium (IMDM).

6. The chemically defined universal medium according to any preceding claim, wherein the basal medium does not comprise serum, lipids, proteins, iron, or growth factors.

7. The chemically defined universal medium according to any preceding claim, wherein the second basal medium comprises Ham's F12.

8. The chemically defined universal medium according to any preceding claim, wherein the polymer comprises or consists of poly(vinyl alcohol) (PVA).

9. The chemically defined universal medium according to any preceding claim, wherein the lipids are a chemically defined lipid mixture optionally comprising one or more, or all of, arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, pluronic F-68, stearic acid, and polysorbate 80.

10. The chemically defined universal medium according to any preceding claim, wherein the activator of the ERK / MAPK pathway comprises an insulin receptor (IR) agonist.

11. The chemically defined universal medium according to any preceding claim, wherein the iron carrier comprises transferrin.

12. The chemically defined universal medium according to any preceding claim, wherein the cell growth and proliferation promoter comprises one of the agents selected from ethanolamine, carbinoxamine, clemastine, dimenhydrinate, chlorphenoxamine, diphenhydramine and doxylamine, or combinations thereof.

13. The chemically defined universal medium according to any preceding claim, wherein the antioxidant comprises vitamin A, vitamin C, vitamin E, monothioglycerol or 2-mercaptoethanol, or any combination thereof.

14. The chemically defined universal medium accordingto any preceding claim, wherein the chemically defined medium (CDUM) does not comprise albumin.

15. Use of the chemically defined universal medium according to any preceding claim for the differentiation of iPSCs, such as human induced pluripotent stem cells (hiPSCs), into a different cell type, wherein the chemically defined universal medium further comprises a differentiation factor selected from a BMP pathway activator, such as BMP4; an activator of SMAD2 / 3 signalling, such asActivin A; a Wnt signalling activator, such as CHIR99021 or Wnt3A; a PI3K inhibitor, such as LY294002; an activator of VEGF signalling, such as VEGF; an activator of FGF signalling, such as FGF2; an activator of PKA signalling, such as cAMP lor 8Bro-cAMP; an activator of EGF signalling, such as EGF; an insulin receptor agonist, such as insulin; a 0-adrenergic agonist, such as isoprenaline; a corticosteroid, such ashydrocortisone or DEXA; a TGF-Beta superfamily inhibitor, such as SB431542; a c-kit receptor (CD117) agonist, such as SCF; an IL-3 receptor agonist, such as IL-3; an IL-4 receptor agonist, such as IL-4; an IL-6 receptor agonist and / or GP130 agonist, such as IL-6 or One M; a TPO receptor agonist, such as TPO; a c-Fms receptor agonist, such as M-CSF; a GM-CSF receptor agonist, such as GM-CSF; an IFNAR agonist, such as IFN-gamma; an immunogen, such as LPS; a BMP pathway inhibitor, such as dorsomorphin; an agent to potentiate Wnt signalling, such as R-Spondinl; a retinoic acid receptor agonist, such as retinoic acid; an FGF receptor agonist, such as FGF10; an FGFR2II IB receptor agonist, such as FGF7; a phosphodiesterase inhibitor, such as IBMX; an extracellular matrix (ECM) growth factor cocktail, such as Matrigel™; one or more inhibitors of Wnt signalling, such as KY0211 and XAV939; a hepatocyte growth factor (HGF) receptor agonist, such as HGF; and a stem cell survival supplement, such as CloneR™ 2.

16. A mesoderm induction medium, wherein the mesoderm induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) a BMP pathway activator, such as BMP4, ii) an activator of SMAD2 / 3 signalling, such as Activin A, iii) a Wnt signalling activator, such as CHIR99021, and iv) a PI3K inhibitor, such as LY294002.

17. An endothelial cell induction medium, wherein the endothelial cell induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an activator of VEGF signalling, such as VEGF ii) an activator of FGF signalling, such as FGF2, and iii) an activator of PKA signalling, such as cAMP or 8Bro-cAMP.

18. A fibroblast induction medium, wherein the fibroblast induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an activator of EGF signalling, such as EGF, ii) an insulin receptor agonist, such as insulin. iii) a BMP pathway activator, such as BMP4, iv) a p-adrenergic agonist, such as isoprenaline, andv) a corticosteroid, such as Hydrocortisone.

19. A hemogenic endothelium induction medium, wherein the hemogenic endothelium induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an activator of VEGF signalling, such as VEGF, ii) an activator of FGF signalling, such as FGF2, iii) a TGF-Beta superfamily inhibitor, such as SB431542, and iv) a c-kit receptor (CD117) agonist, such as SCF.

20. A hematopoietic cell induction medium, wherein the hematopoietic cell induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an activator of VEGF signalling, such as VEGF, ii) an activator of FGF signalling, such as FGF2, iii) c-kit receptor (CD117) agonist, such as SCF, iv) an IL-3 receptor agonist, such as IL-3, v) an IL-6 receptor agonist and / or GP130 agonist, such as IL-6, and vi) a TRO receptor agonist, such as TRO.

21. A monocyte induction medium, wherein the monocyte induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an IL-3 receptor agonist, such as IL-3, ii) an IL-6 receptor agonist and / or GP130 agonist, such as IL-6, and iii) a c-Fms receptor agonist, such as M-CSF.

22. A macrophage induction medium, wherein the macrophage induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises a c-Fms receptor agonist, such as M-CSF (MacrophageColony-Stimulating Factor).

23. A macrophage polarisation medium MO, wherein the polarisation medium MO comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises a c-Fms receptor agonist, such as M-CSF.

24. A macrophage polarisation medium Ml, wherein the polarisation medium Ml comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an IFNAR agonist, such as IFN-gamma, and ii) an immunogen, such as LPS.

25. A macrophage polarisation medium M2, wherein the polarisation medium M2 comprises the chemically defined universal medium according any one of claims 1-14, and wherein the chemically defined universal medium further comprises an IL-4 receptor agonist, such as IL-4.

26. An immature dendritic cell induction medium, wherein the immature dendritic cell induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) a GM-CSF receptor agonist, such as GM-CSF, and ii) an IL-4 receptor agonist, such as IL-4.

27. A mature dendritic cell induction medium, wherein the mature dendritic cell induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an IFNAR agonist, such as TNF-alpha, and ii) an immunogen, such as LPS.

28. An endoderm induction medium, wherein the endoderm induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) a Wnt signalling activator, such as CHIR99021, ii) a PI3K inhibitor, such as LY294002, and iii) an activator of SMAD2 / 3 signalling, such as Activin A.

29. An anterior foregut endoderm induction medium, wherein the anterior foregut endoderm induction medium comprises the chemically defined universal medium according to any one of claims1-14, and wherein the chemically defined universal medium further comprises: i) a BMP pathway inhibitor, such as dorsomorphin, and ii) a TGF-Beta superfamily inhibitor, such as SB431542.

30. A lung progenitor cell induction medium, wherein the lung progenitor cell induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) a Wnt signalling activator, such as Wnt3A, ii) an agent to potentiate Wnt signalling, such as R-Spondinl, iii) a BMP pathway activator, such as BMP4, and iv) a retinoic acid receptor agonist, such as retinoic acid.

31. A distal lung maturation induction medium, wherein the distal lung maturation induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) a Wnt signalling activator, such as CHIR99021, ii) an FGF receptor agonist, such as FGF1O, iii) an FGFR2II I B receptor agonist, such as FGF7, iv) a phosphodiesterase inhibitor, such as IBMX, v) an activator of PKA signalling, such as cAMP, and vi) a corticosteroid, such as DEXA.

32. A foregut induction medium, wherein the foregut induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises an activator of SMAD2 / 3 signalling, such as Activin A.

33. A hepatic endoderm induction medium, wherein the hepatic endoderm induction medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) a TGF-Beta superfamily inhibitor, such as SB431542, ii) a BMP pathway activator, such as BMP4, and iii) a FGF receptor agonist, such as FGF10.

34. A hepatic maturation medium, wherein the hepatic maturation medium comprises the chemically defined universal medium according to any one of claims 1-14, and wherein the chemically defined universal medium further comprises: i) an IL-6 receptor agonist and / or GP130 agonist, such as oncostatin M (One M, orOSM), and ii) a hepatocyte growth factor (HGF) receptor agonist, such as HGF.

35. A cardiomyocyte (CMC) induction medium, wherein the CMC induction medium comprises the chemically defined universal medium according to any of claims 1-14, and wherein the chemically defined universal medium further comprises: i) a BMP pathway activator, such as BMP4, ii) an activator of SMAD2 / 3 signalling, such as Activin A, iii) one or more inhibitors of Wnt signalling, such as KY0211 and XAV939, and iv) optionally an extracellular matrix (ECM) growth factor cocktail.

36. A method for differentiating induced pluripotent stem cells (iPSCs) into mesodermal progenitor cells, the method comprising:1) providing induced pluripotent stem cells (iPSCs);2) inducing differentiation of the iPSCs into mesodermal progenitor cells by culturing the iPSCs in a mesoderm induction medium according to claim 16; and3) optionally further differentiating the into a different cell type.

37. A method of differentiating mesodermal progenitor cells into endothelial cells, the method comprising the steps of: providing mesoderm progenitor cells, optionally in accordance with the method of claim 36; and incubating the mesoderm progenitor cells in an endothelial cell induction medium according to claim 17; and optionally assembling the endothelial cells into an organoid.

38. A method of differentiating mesodermal progenitor cells into fibroblasts, the method comprising the steps of:providing mesoderm progenitor cells, optionally in accordance with the method of claim 36; and incubating the mesoderm progenitor cells in fibroblast induction medium according to claim18; and optionally assembling the fibroblasts into an organoid.

39. A method of differentiating mesodermal progenitor cells into hemogenic endothelium, the method comprising the steps of: providing mesodermal progenitor cells, optionally in accordance with the method of claim 35; and incubating the mesoderm progenitor cells in hemogenic endothelium induction medium according to claim 19.

40. A method of differentiating hemogenic endothelium into hematopoietic cells, the method comprising the steps of: providing hemogenic endothelium cells, optionally in accordance with the method of claim39; and incubating the hemogenic endothelium cells in a hematopoietic cell induction medium according to claim 20; and optionally further differentiating the hematopoietic cells to form a different cell type.

41. A method of differentiating hematopoietic cells into T cells, the method comprising the steps of: providing hematopoietic cells, optionally in accordance with the method of claim 40; and culturing the hematopoietic cells on a layer of feeder cells, such as OP9-DL1 cells, while incubating the hematopoietic cells in a T cell induction medium, wherein the T cell induction medium comprises: i) a basal medium, such as MEM a, ii) animal serum, such as foetal bovine serum, iii) an anti-oxidant, such as monothioglycerol, iv) a c-kit receptor agonist, such as SCF, v) an IL-7 receptor agonist, such as IL-7, vi) a TPO (thrombopoietin) receptor agonist, such as TPO, and vii) a FLT3 agonist, such as FLT3L.

42. A method of differentiating hematopoietic cells into monocytes, the method comprising the steps of: providing hematopoietic cells, optionally in accordance with the method of claim 40; and incubating the hematopoietic cells in the monocyte induction medium according to claim 21.

43. A method of differentiating monocytes into macrophages, the method comprising the steps of: providing monocytes, optionally in accordance with the method of claim 42; and incubating the monocytes in the macrophage induction medium according to claim 22; and optionally polarising the macrophages; and optionally assembling the macrophages into an organoid.

44. A method of polarising macrophages, the method comprising the steps of: providing macrophages, optionally in accordance with the method of claim 43; and incubating the macrophages in the polarisation medium MO according to claim 23, polarisation medium Ml according to claim 24, or polarisation medium M2 according to claim25; and optionally assembling the polarised macrophages into an organoid.

45. A method of differentiating monocytes into immature dendritic cells, the method comprising the steps of: providing monocytes, optionally in accordance with the method of claim 42; and incubating the monocytes in the immature dendritic cell induction medium according to claim26.

46. A method of differentiating immature dendritic cells into mature dendritic cells, the method comprising the steps of: providing immature dendritic cells, optionally in accordance with the method of claim 45; and incubating the immature dendritic cells in the mature dendritic cell induction medium according to claim T); and optionally assembling the mature dendritic cells into an organoid.

47. A method of differentiating induced pluripotent stem cells (iPSCs) into endoderm, the method comprising the steps of:1) providing induced pluripotent stem cells (iPSCs);2) inducing differentiation of the iPSCs into endoderm by culturing the iPSCs in the endoderm induction medium according to claim 28.

48. A method of differentiating endoderm into anterior foregut endoderm, the method comprising the steps of: providing endoderm, optionally in accordance with the method of claim 47; and incubating the endoderm in the anterior foregut endoderm induction medium according to claim 29.

49. A method of differentiating anterior foregut endoderm cells into lung progenitor cells, the method comprising the steps of: providing anterior foregut endoderm cells, optionally in accordance with the method of claim48; and incubating the anterior foregut endoderm cells in the lung progenitor cell induction medium according to claim 30.

50. A method of differentiating lung progenitor cells into distal lung cells, such as alveolar epithelial type II cells, the method comprising the steps of: providing lung progenitor cells, optionally in accordance with the method of claim 49; and incubating the lung progenitor cells in the distal lung maturation induction medium according to claim 31; and optionally assembling the distal lung cells into an organoid.

51. A method of differentiating endoderm into foregut endoderm, the method comprising the steps of: providing endoderm, optionally in accordance with the method of claim 47; and incubating the endoderm in the foregut induction medium according to claim 32.

52. A method of differentiating foregut endoderm into hepatic endoderm, the method comprising the steps of: providing foregut endoderm, optionally in accordance with the method of claim 51; incubating the foregut endoderm in the hepatic endoderm induction medium according to claim 33.

53. A method of differentiating hepatic endoderm into hepatocytes, the method comprising the steps of: providing hepatic endoderm, optionally in accordance with the method of claim 52; incubating the hepatic endoderm in the hepatic maturation medium according to claim 34.

54. A method of differentiating induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMCs), the method comprising: providing iPSCs; incubating the iPSCs in a CMC induction medium in accordance with claim 35 to obtain immature CMCs; and incubating the immature CMCs in a CMC maturation medium.

55. An organoid comprising: endothelial cells formed according to the method of claim 37; fibroblasts formed according to the method of claim 38; macrophages or polarised macrophages formed according to the method of claims 43 or 44; mature dendritic cells formed according to the method of claim 46; or distal lung cells formed according to the method of claim 50; or combinations thereof.

56. An organoid comprising: macrophages or polarised formed according to the method of claims 43 or 44; mature dendritic cells formed according to the method of claim 46; fibroblasts formed according to the method of claim 38; endothelial cells formed according to the method of claim 37; and / or hepatocytes according to the method of claim 53; or combinations thereof.

57. An organoid comprising cardiomyocytes according to the method of claim 54.

58. A disease model, wherein the disease model comprises an organoid according to any of claims 55-57, wherein the organoid has a genetic mutation associated with a disease, and / or the organoid has been treated with an agent to induce a disease state in the organoid.

59. The use of the disease model according to claim 58 to identify agents capable of preventing or treating the disease, wherein the organoid is treated with a potential agent before, during or after the organoid is treated with a disease-inducing agent.

60. A method of screening for agents capable of preventing or treating a disease, the method using the organoids according to any of claims 55-57, wherein the organoid is genetically manipulated or treated with a potential agent before, during or after the organoid is treated to induce disease; and determining if the potential agent or genetic target has any effect in inhibiting or preventing the development of disease in the organoid, or the reduction in disease after it has developed in the organoid.

61. A method for seeding of an organoid with cells from a donor, and tracking the cells to assay one or more of survival, proliferation and isolation of engrafted cells, wherein the organoid is according to any of claim 55-57.

62. A method for producing a product from an organoid according to any of claims 55-57, the method comprising the incubation of the organoid in vitro, and harvesting the product produced from the organoid.

63. A method of screening for biomarkers of a disorder in an organ or tissue, the method comprising the monitoring of biomarkers released from an organoid according to any of claims 55-57, or cells engrafted therein, or biomarkers in tissue or cellular extracts of an organoid according to any of claims55-57, or released in the medium that is and / or has been in contact with the organoid.

64. A kit comprising two or more of the cell culture mediums according to claims 1-14 or 16-35.

65. Cells obtained by a method according to any of claims 36 to 54, or harvested from any of the organoids according to any of claims 55-57.

66. An assembloid comprising two or more organoids, wherein at least one organoid is according to any of claims 55-57.

67. A method of generating an assembloid, the method comprising incubating two or more different organoids, wherein at least one of the organoids comprises an organoid according to any of claims 55-57.

68. Cells obtained by a method according to claims 67, or harvested from the assembloid according to claim 66.

69. A method of screening for biomarkers of a disorder in an organ or tissue, the method comprising the monitoring of biomarkers released from an assembloid according to claim 66, or cells engrafted therein, or biomarkers in tissue or cellular extracts of an assembloid according to claim 66, or released in the medium that is and / or has been in contact with the assembloid.

70. Use of the chemically defined universal medium according to any one of claims 1-14 for the isolation and / or culture of mammalian cells.

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