Methods of pluripotent stem cell expansion

Abstract

The invention relates to methods of pluripotent stem cell (PSC) culture, PSC cultured in 3D suspension culture bioreactor systems, expansion of PSC in 3D suspension, and differentiation of 3D expanded PSC clusters into Definitive Endoderm (DE).

Description

[0001] METHODS OF PLURIPOTENT STEM CELL EXPANSION

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods of pluripotent stem cell (PSC) culture, PSC cultured in 3D suspension culture bioreactor systems, expansion of PSC in 3D suspension, and differentiation of 3D expanded PSC clusters into Definitive Endoderm (DE).

[0004] Background of the invention

[0005] Large scale expansion of pluripotent stem cells (PSC), such as induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs), is essential to provide starting material for manufacturing of many commercially important cell therapy products, but also for other applications, such as cell expansion for drug screening purposes. While pluripotent stem cells are typically cultured on 2D culture surfaces coated with suitable matrices, and in the presence of pluripotency-maintaining media and supplements, this requires large culture areas and tissue culture material if larger amounts of pluripotent cells are needed.

[0006] Alternatively, they can also be expanded in the same or similar media in agitated suspension culture as pluripotent cell aggregates (3D expansion / 3D suspension culture) to supply large amounts of pluripotent cells in an efficient and scalable manner.

[0007] In cell therapy, this is most relevant for all cell types that require large cell numbers per dose, for example pancreatic islet cells, lymphocytes, cardiomyocytes. Due to limited proliferation potential during or at the final stages of most of differentiation procedures, it is essential to supply large amounts of fully differentiation-competent pluripotent cells as starting material. Therefore, systems and methods for the scalable / large-scale suspension culture expansion of fully differentiation competent pluripotent PSCs are of high interest.

[0008] In general, inefficient DE differentiation after 3D expansion in bioreactors presents a problem for upscaling of PSC-based cell therapy manufacturing, at pre-clinical stage, during manufacturing of clinical trial material and especially during manufacturing of market material.

[0009] DE differentiation typically starts with dissociation of cells, which involves medium exchange and disturbance of the culture (e.g. Sahabian et al. 2019, Cells (8)1571). Problems with inefficient DE differentiation after 3D expansion are relevant for all cell types that are derived from definitive endoderm (e.g. islet like clusters, liver, lung, oesophagus, thyroid), but may also affect mesodermal cells (e.g. cardiac cells, lymphocytes, skeletal muscle, endothelial cells) which initially need to pass through the mesendoderm stage common to both mesodermal and definitive endoderm cells.

[0010] Hence, there is a need for culturing methods that allow for fold expansion of PSCs over several days in large-scale 3D suspension culture while retaining a high percentage of DE differentiation potential of the PSCs.

[0011] Objectives and Summary of the Invention

[0012] Initiation of differentiation by direct cluster transfer of 3D-expanded PSC clusters is especially attractive, since there are no dissociation and re-seeding steps and associated cell losses.

[0013] Direct cluster transfer also facilitates maintaining a closed process in the context of GMP manufacturing, a key advantage with regard to obtaining a cost-effective, safe and regulatory rules-compliant manufacturing process.

[0014] However, the inventors have surprisingly found that on or after the third day of 3D suspension expansion of non-differentiated PSCs, the ability to form Definitive Endoderm (DE, first step of beta / islet cell differentiation) from directly seeded / transferred PSC clusters strongly declines, despite persistently high expression of all analyzed pluripotency markers. This observed PSC phenotype is subsequently called “DE-differentiation-defective state”.

[0015] The long-term 3D expanded PSC clusters do not lose DE-differentiation capacity completely. However, for industrial manufacturing of therapeutically relevant cells from PSCs, DE percentages of more than 90%, ideally 95% or higher are essential, in order to avoid the generation of unsafe end product (e.g. due to residual pluripotent iPSCs), low efficiency processes (generation of too much unwanted non-endodermal cell type contaminants), and / or variability in manufacturing process performance.

[0016] Furthermore, the inventors have surprisingly found that a characteristic molecular signature can be assigned to the DE-differentiation-defective state. By comparing gene and protein expression in DE-differentiation competent vs. defective long-term 3D expanded PSC clusters, several deregulated pathways and marker genes could be identified that can serve as a tool for the detection and quantification of the presence of DE-differentiation-defective cell states during PSC expansion and differentiated cell manufacturing. The inventors surprisingly found that increasing stirring speed in 3D suspension cultures increased or restored the capacity of PSCs to differentiate into DE by direct cluster transfer after more than 2 days in culture to over 90%, hence avoiding the DE-differentiation defect. This was particularly surprising since higher stirring speeds are usually associated with increased shear forces, which lead to shear stress, which in turn can affect PSC expansion and differentiation capacity.

[0017] It was also surprisingly found that efficient DE differentiation from long-term (> 2 days) 3D expanded pluripotent iPSC clusters can be initiated by direct cluster transfer (without dissociation and re-aggregation prior to or at the start of differentiation), and that novel cell populations able to give rise to high efficiency DE differentiation after direct cluster transfer can be generated using properly chosen conditions and / or by adding selected small molecule compounds.

[0018] Hence, in a first aspect, the invention provides a method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and differentiating expanded PSCs into DE, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3; c) culturing PSC clusters for at least 2 days; and d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters.

[0019] In one embodiment, the PSC are expanded by at least 1.5 fold by day 4 of culture compared to the starting material. In one preferred embodiment, the PSC are expanded by at least 8 fold by day 4 of culture compared to the starting material.

[0020] In one embodiment, the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a volumetric power input of less than 0.4 W / m3. In one embodiment, improved differentiation potential means that at least 90% of PSC are capable of differentiating into DE. Preferably, at least 93% of PSC are capable of differentiating into DE. More preferably, at least 95% of PSC are capable of differentiating into DE.

[0021] In one embodiment, the culture volume is between 0.1 L and 2000L. The skilled person is aware that the stirring speed (revolutions per minute, rpm) must be adapted to the volume of the culture.

[0022] In one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm.

[0023] In one embodiment, the volumetric power input is between > 0.4 W / m3to < 2.5 W / m3. Hence, for each culture volume, the rpm must be adapted accordingly.

[0024] Thus, in one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2.5 W / m3.

[0025] In one embodiment, the culture volume is about 0.1 L and the stirring speed is between > 80 rpm to <150 rpm.

[0026] In one embodiment, the culture volume is about 0.23 L and the stirring speed is between > 75 rpm to <135 rpm.

[0027] In one embodiment, the culture volume is about 1.5 L and the stirring speed is between > 73 rpm to < 110 rpm.

[0028] In one embodiment, the culture volume is about 10 L and the stirring speed is between > 50 rpm to < 95 rpm.

[0029] In one embodiment, the culture volume is about 50 L and the stirring speed is between > 45 rpm to < 85 rpm.

[0030] In one embodiment, the culture volume is about 100 L and the stirring speed is between > 40 rpm to < 80 rpm.

[0031] In one embodiment, the culture volume is about 500 L and the stirring speed is between > 35 rpm to < 70 rpm. In one embodiment, the culture volume is about 1000 L and the stirring speed is between > 30 rpm to < 65 rpm.

[0032] In one embodiment, the culture volume is about 2000 L and the stirring speed is between > 25 rpm to < 60 rpm.

[0033] The volumetric power input is determined using the formula Equation (I): wherein P = Power [W],

[0034] V = liquid volume [m3],

[0035] N = impeller rotational speed [s’1],

[0036] D = impeller diameter [m], w = impeller width [m], bn= number blades of impeller [-],

[0037] Dv = diameter of bioreactor [m],

[0038] 0 = blade angle [°], p = liquid density [kg / m3], wherein the liquid density is determined for the cell-free fraction of the liquid contained in the bioreactor.

[0039] In one embodiment, the bioreactor is a stirred-tank bioreactor.

[0040] In one embodiment, the PSC are human iPSC or human ESC.

[0041] In a second aspect, the invention also provides a population of PSCs capable of differentiating into Definitive Endoderm (DE) obtained by the method described above.

[0042] In one embodiment, at least 90% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after 4 days in culture, wherein the PSC clusters are not dissociated before differentiation. In a third aspect, the invention also provides a population of Definitive Endoderm cells produced by direct cluster transfer differentiation of the population of PSC described above.

[0043] In another aspect, the invention provides a method of PSC differentiation into Definitive Endoderm, the method comprising a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) culturing the PSC for at least 3 days; c) inducing PSC clusters to differentiate to DE by direct cluster transfer differentiation, wherein the method does not comprise a step of dissociation of PSC clusters before differentiation into DE.

[0044] The inventors also surprisingly found that the DE-differentiation defective state can be overcome by treating DE-differentiation-defective PSC clusters with Blebbistatin.

[0045] Hence, in a further aspect, the invention also provides a method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and differentiating expanded PSCs into DE, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) treating the PSC clusters with Blebbistatin; c) culturing PSC clusters for at least 2 days; and d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation.

[0046] In one embodiment, the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters.

[0047] In one embodiment, at least 90% of expanded PSCs are capable of differentiating into DE.

[0048] In one embodiment, the PSC clusters are treated with 5-15 pM Blebbistatin. In a preferred embodiment, the PSC clusters are treated with 10 pM Blebbistatin. In one embodiment, the PSC clusters are treated with 10 pM Blebbistatin for at least 24 hours. In one embodiment, the method further comprises modulating the stirring speed between > 35 rpm to <150 rpm. In one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm.

[0049] In one embodiment, the volumetric power input is between > 0.4 W / m3to < 2.5 W / m3. Hence, for each culture volume, the rpm must be adapted accordingly.

[0050] Thus, in one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2.5 W / m3.

[0051] In a preferred embodiment, the culture volume is between 200ml and 250ml, such as 230ml; and the stirring speed is between 65 rpm and 90 rpm.

[0052] The inventors also surprisingly found that the DE-differentiation-defective state of PSC clusters can be overcome by modulating the type or concentration of bFGF that is added to the culture during expansion. Usually, during the PSC 3D suspension expansion stage, a 146 amino acid variant of bFGF is added to the culture. However, the inventors found that using a 154 amino acid variant of bFGF can increase the percentage of DE-differentiation- capable PSCs to at least 90%. Furthermore, increasing the concentration of 146 amino acid bFGF 6-fold from 50 ng / mL to 300 ng / mL provided a similar effect.

[0053] Hence, in a further aspect, the invention provides a method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and differentiating expanded PSCs into DE, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days in culture medium, wherein bFGF signalling is modulated during culturing; and c) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters; and wherein at least 90% of PSCs are capable of differentiating into DE. Modulation bFGF signalling means that a specific bFGF variant is added to the culture. Dosage and variant of bFGF can influence the culture. In one embodiment, the culture medium comprises 100 - 500 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 , preferably 300 ng / mL. In another embodiment, the culture medium comprises 1 - 100 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2, preferably 50ng / mL.

[0054] In one embodiment, the method further comprises modulating the stirring speed between > 35 rpm to <150 rpm. In one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm.

[0055] In one embodiment, the volumetric power input is between > 0.4 W / m3to < 2.5 W / m3. Hence, for each culture volume, the rpm must be adapted accordingly.

[0056] Thus, in one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2.5 W / m3.

[0057] In a preferred embodiment, the culture volume is between 200ml and 250ml, such as 230ml; and the stirring speed is between 65 rpm and 90 rpm.

[0058] The inventors have further characterized populations of PSCs that have lost the capacity to differentiate into Definitive Endoderm with rates of at least 90%. These populations of PSCs are termed “DE-differentiation-defective”.

[0059] A DE-differentiation-defective status in 3D-expanded PSC clusters can be determined by quantifying the expression of KDR, CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, S100A4 and SOX21 . Thus, the inventors have, for the first time, described a new population of PSCs which is DE-differentiation-defective, but can be rescued and efficiently differentiated into DE, with at least 90% of cell differentiating into DE by direct cluster transfer using the methods described herein. These methods include one or more of increasing stirring speed, adding Blebbistatin, and modulating bFGF signalling.

[0060] Hence, in a further aspect, the invention provides a population of DE-differentiation-defective PSCs, wherein the population of PSCs expresses one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, S100A4 and SOX21 , or any combination thereof at higher levels than a population of DE-competent PSCs. Likewise, the invention provides a population of DE-differentiation-competent PSCs, wherein the population of PSCs expresses one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21, or any combination thereof at lower levels than a population of DE-competent PSCs.

[0061] DE-competence is defined as at least 90% of PSCs of a population of PSCs being capable of differentiating into DE. In one embodiment, the population of PSCs comprises at least one cluster of PSCs.

[0062] In one embodiment, the lower or higher expression level means expression below or above a threshold. The threshold is determined by comparing marker expression in distinct DE- differentiation-defective and DE-differentiation-capable populations of PSCs.

[0063] Hence, in another aspect, the invention also relates to a method of detecting DE- differentiation-defective PSCs clusters, the method comprising the steps of a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days; and c) quantifying the expression of one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21.

[0064] In one embodiment, quantifying the expression of one or more markers further comprises comparing the expression level of the one or more markers with a threshold or control value.

[0065] In another aspect, the invention also relates to a population of PSCs obtained by a) seeding iPSC in a bioreactor to initiate 3D suspension culture under conditions that allow iPSC cluster formation; b) culturing iPSC clusters for at least 2 days; and c) inducing iPSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of iPSC clusters; and wherein at least 90% of PSCs have the capacity to differentiate into DE; wherein the PSCs express low levels of one or more markers selected from the group consisting of CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, KDR, S100A4 and SOX21.

[0066] In one embodiment, differentiation into DE is determined by positive expression of CXCR4 and EPCAM.

[0067] In one embodiment, low level of marker expression means expression below a threshold. The threshold is determined by comparing marker expression in distinct DE-differentiation- defective and DE-differentiation-capable populations of PSCs.

[0068] In one embodiment, the PSCs are human induced PSCs or human ESCs. Preferably, the PSCs are induced pluripotent stem cells (iPSCs).

[0069] Figure Legends

[0070] Figure 1 : Experimental outline of stirring speed experiments. Pluripotent stem cells were expanded in stirred tank bioreactors at three different speeds for four days. After days 2, 3 and 4, PSC clusters were taken out of the bioreactors and transferred into six well plate for differentiation towards DE.

[0071] Figure 2: DE differentiation outcome at different stirring speeds. Flow cytometry assessment of EPCAM / CXCR4 of 2, 3 and 4 day expanded iPSC clusters in the DASGIP® Bioblock bioreactor system at 500 mL culture volume and given stirring speeds with a 8- blade impeller. Subsequent differentiation was conducted in 6 well plates. d2, d3, d4: day 2, day 3, day 4; rpm: revolutions per minute, DE: definitive endoderm.

[0072] Figure 3: DE differentiation outcome at different stirring speeds. Flow cytometry assessment of EPCAM / CXCR4) of expanded iPSC clusters in the Applikon MiniBio 500 bioreactor system at 230 mL culture volume and given stirring speeds with a 3-blade impeller. Subsequent differentiation was conducted in 6 well plates. A: 2, 3 and 4 day expanded iPSC clusters cultured at stirring speeds of 67rpm, 89 rpm and 112 rpm. d2, d3, d4: day 2, day 3, day 4; rpm: revolutions per minute, DE: definitive endoderm. B: 2 and 3 day expanded iPSC clusters cultured at stirring speeds of 67rpm, 75rpm, 82rpm, 89rpm and 112rpm.

[0073] Figure 4: DE differentiation outcome at different stirring speeds. Flow cytometry assessment of EPCAM / CXCR4 of 2, 3 and 4 day expanded iPSC clusters in the Applikon Bio 3L bioreactor system at 1500 mL culture volume and given stirring speeds with a 3-blade impeller. Subsequent differentiation was conducted in 6 well plates. d2, d3, d4: day 2, day 3, day 4; rpm: revolutions per minute, DE: Definitive Endoderm.

[0074] Figure 5: DE differentiation with Blebbistatin treatment. Pluripotent stem cells were expanded in stirred tank bioreactors at slow speeds for four days (d4). After day three (d3), PSC clusters were taken out of the bioreactors and transferred into six well plate where they were either treated with 10 pM Blebbistatin or in control (Ctrl) conditions (0.09% DMSO) for 24 h. Afterwards clusters were transferred to differentiation towards Definitive Endoderm (DE).

[0075] Figure 6: DE differentiation with different bFGF lengths and concentrations. Pluripotent stem cells were expanded in stirred tank bioreactors at 75 rpm and 230 mL for three days. In the bioreactor phase the amino acid chain length of bFGF (146 amino acid vs 154 amino acid) as well as the concentration of the 146 amino acid bFGF (1x vs 6x) was modulated. After day three (d3), PSC clusters were taken out of the bioreactors and transferred into six well plate for differentiation towards Definitive Endoderm (DE).

[0076] Figure 7: Characterization of DE defective cells by qPCR. A: Quantification of identified target genes (KDR, CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, S100A4 and SOX21) with quantitative PCR (qPCR) in DE competent and DE defective iPSC clusters. B: Fold upregulation of marker genes of DE defective iPSC clusters compared to DE competent iPSC clusters.

[0077] DETAILED DESCRIPTION OF THE INVENTION

[0078] Definitions

[0079] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0080] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0081] The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.

[0082] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0083] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a compound is defined to be obtainable from a specific source, this is also to be understood to disclose a compound which is obtained from this source.

[0084] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

[0085] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0086] A “pluripotent stem cell” or “PSC” as used herein refers to a stem cell that has the potential to differentiate into any of the three germ layers, i.e. endoderm, mesoderm or ectoderm, but not into extra-embryonic tissues, i.e. placenta or yolk sac. The term “PSC” or “PSCs” as used herein generally refers to a plurality of cells unless it is explicitly stated that a single cell is meant. Markers for undifferentiated PSC include OCT4 (POLI5F1 , POU class 5 homeobox 1), NANOG (Nanog homeobox), LIN28A (lin-28 homolog A), ESRG (embryonic stem cell related), SOX2 (SRY-box transcription factor 2), ALPL (alkaline phosphatase, biomineralization associated), SSEA3 (Stage-specific embryonic antigen 3), SSEA4 (Stagespecific embryonic antigen 4), TRA-1-60 (Tumor rejection antigen 1-60) and TRA-1-81 (Tumor rejection antigen 1-81). TRA-1-60 and TRA-1-81 are monoclonal antibodies that recognize specific carbohydrate epitopes on the surface of PSCs. SSEA3 and SSEA4 are cell surface glycosphingolipids expressed by stem cells at specific stages of embryonic development.

[0087] The term “induced pluripotent stem cell” or “iPSC” as used herein refers to a type of PSC generated directly from a somatic cell by introduction of pluripotency-associated genes, so- called reprogramming factors, such as OCT4 (POU5F1 , POU class 5 homeobox 1), SOX2 (SRY-box transcription factor 2), KLF4 (Kruppel-like factor 4) and MYC (MYC protooncogene, bHLH transcription factor).

[0088] The term “embryonic stem cell” or “hESC” as used herein refer to PSCs derived from the inner cell mass of a blastocyst of an early mammalian embryo.

[0089] The term “differentiation capacity” or “differentiation potential” as used herein refers to the ability of a stem cell to differentiate into a specific cell type. When carrying out differentiation of a population of PSC, a certain fraction will differentiate into the chosen cell type, while another fraction will not. Thus, it is possible to refer to the differentiation capacity of a population of PSCs with percentages.

[0090] A “Definitive Endoderm” (DE) cell as used herein is a cell at a stage between a pluripotent cell and an endodermal tissue cell, such as a gut tube cell, thyroid cell, pancreatic cell, or hepatic cell. It can be characterized by the expression of S0X17 (SRY-box transcription factor 17), F0XA2 (forkhead box A2), a combination of CXCR4 (C-X-C motif chemokine receptor 4) and EPCAM (epithelial cell adhesion molecule), or a combination of CXCR4 and c-KIT (KIT proto-oncogene, receptor tyrosine kinase).

[0091] The term “3D suspension culture” refers to a culture wherein cells or cell clusters are cultured in a volume of medium under continuous agitation, as opposed to a 2D cell culture wherein cells adhere to a surface, such as a coated cell culture dish, a support matrix or feeder cell layer. 3D suspension cultures are initiated by dissociated of a 2D culture, followed by seeding of small clumps of cells or single cells derived from 2D adherent cultures into the 3D culture medium, such as seeding medium. An expansion phase in which cells are multiplied follows, which usually involves growth medium. 3D suspension cultures may be passaged to control cell cluster size. For passaging, cell clusters may be dissociated into smaller clusters or single cells.

[0092] The terms PSC “cluster” or “aggregate”, as used interchangeable herein, refers to the natural accumulation of undifferentiated PSCs in 3D suspension culture without carriers, scaffolds or encapsulation.

[0093] The terms “direct cluster differentiation” or “direct cluster transfer” refer to a method of differentiation of PSC wherein the PSC are differentiated as clusters, in contrast to differentiation methods that start with a step of dissociation of PSC clusters into single cells before re-aggregation and initiating differentiation. Commonly used DE-differentiation methods employ the steps of PSC cluster dissociation followed by reaggregation, before differentiation. Direct cluster transfer differentiation differs in that PSC clusters are not dissociated or dissociated and reaggregated before differentiation. Dissociation of cells may be performed at other stages of the culturing method that are not related to differentiation, for example before seeding of the 3D suspension culture, during passaging, before cryopreservation of intermediate or end stages, or to select certain subpopulations during intermediate or end stages.

[0094] The term “expression level” refers to the levels or mRNA or of protein of a specific gene, e.g. a marker gene. Methods of determining mRNA or protein levels are known in the art.

[0095] Preferably, mRNA expression levels are determined by quantitative PCR. Protein levels may be determined by e.g. Western Blot. The term “lower expression level” or “higher expression level” as used herein refers to the expression level of one or more markers in a cell population compared to the expression level of the same markers in another cell population, or compared to a threshold level. A threshold level can be determined by comparing the expression levels of one or more markers in cell populations with known phenotypes, for example, as herein, DE-differentiation-defective or DE-differentiation-capable cells.

[0096] Culture method

[0097] The invention relates to methods of 3D suspension culture of PSCs, such as iPSC or hPSC.

[0098] 3D suspension culture of PSCs leads to the formation of small cell aggregates, i.e. PSC clusters. Differentiation protocols, especially DE differentiation protocols, commonly start with a step of dissociation, whereby the PSC clusters are separated into single cells and reseeded before differentiation media and / or differentiation factors are added.

[0099] The inventors have developed a method to differentiate clusters of PSCs directly, without a step of dissociation into single cells.

[0100] Hence, the invention provides a method of culturing PSC in a 3D suspension culture, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation of step d) does not comprise dissociation of PSC clusters. Furthermore, the invention provides a method of differentiating PSCs into Definitive Endoderm cells, comprising the steps of: a) seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation of step d) does not comprise dissociation of PSC clusters.

[0101] In one embodiment, direct cluster transfer differentiation comprises the following steps: i) transferring non-dissociated clusters into a new container; ii) allowing cells to settle and removing supernatant; optionally washing with cell culture medium iii) resuspending non-dissociated clusters in differentiation medium.

[0102] In one embodiment, the differentiation medium comprises ROCK Inhibitor, Activin A, BMP4, FGF-2, Heparin, GSK3 inhibitor, and Ascorbic Acid.

[0103] In a preferred embodiment, the differentiation medium comprises 10 pM of ROCK Inhibitor, 100 ng / mL Activin A, 5 ng / mL BMP4, 5ng / mL FGF-2, 10 ng / mL Heparin, 1 pM GSK3 inhibitor, and 0.5 mM Ascorbic Acid.

[0104] When culturing PSC clusters in 3D suspension culture long term, i.e. more than 2 days in continuous culture, PSCs lose the capacity to differentiate into Definitive Endoderm (DE), i.e. they become DE-defective. While it is still possible to differentiate PSC into DE by direct cluster transfer differentiation when some of the cells have become DE-defective, it is of clinical interest to produce large volume cultures of expanded PSC wherein at least 90% of the PSC retain the capacity to differentiate into DE by direct cluster transfer differentiation.

[0105] The inventors surprisingly found that by increasing the stirring speed, they could prevent a large proportion of PSC clusters from becoming DE-defective, thereby enabling expansion of PSC that retain the capacity to differentiate into DE by direct cluster transfer differentiation, i.e. without a dissociation step, after more than 2 days in culture. Hence, the invention relates to a method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and subsequently differentiating expanded PSCs into DE, comprising the steps of:

[0106] • seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation;

[0107] • culturing the PSC clusters for at least 2 days, preferably at least 3 days, more preferably at least 4 days;

[0108] • performing one or more of the following during the culturing:

[0109] (i) providing a source of agitation to the PSC clusters that produces a volumetric power input of at least 0.4 W / m3;

[0110] (ii) treating the PSC clusters with Blebbistatin; and / or

[0111] (iii) modulating bFGF signalling; and

[0112] • inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise dissociation of PSC clusters.

[0113] In one embodiment, the method of the invention comprises a culturing phase, during which PSCs are expanded, and a differentiation phase during which PSCs are differentiated into Definitive Endoderm (DE).

[0114] In a preferred embodiment, the interventions performed during the culturing phase, e.g. agitation, Blebbistatin treatment or modulating bFGF signalling, are not performed during differentiation.

[0115] Hence, in one embodiment, the invention relates to a method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and subsequently differentiating expanded PSCs into DE, comprising the steps of:

[0116] • seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation;

[0117] • culturing the PSC clusters for at least 2 days, preferably at least 3 days, more preferably at least 4 days;

[0118] • performing one or more of the following during the culturing:

[0119] (i) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3, preferably wherein the agitation is continuous; (ii) treating the PSC clusters with Blebbistatin, preferably wherein the PSC clusters are treated with 5-15 pM Blebbistatin; and / or

[0120] (iii) modulating bFGF signalling, preferably wherein modulating bFGF signalling comprises adding 100 to 500 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 ; or adding 1 to 100 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2; and

[0121] • stopping performing (i), (ii), and / or (iii); and

[0122] • inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation; wherein the direct cluster transfer differentiation does not comprise dissociation of PSC clusters.

[0123] In one embodiment, stopping agitation comprises reducing the power input to less than 0.4 0.4 W / m3, preferably reducing the power input to between 0 to less than 0.4 W / m3.

[0124] In one embodiment, stopping Blebbistatin treatment comprises reducing the Blebbistatin concentration in the medium to below 5 pM Blebbistatin. This can be achieved for example by changing the medium, removing the cells from the medium, or through using up the compound over time.

[0125] In one embodiment, stopping modulation of bFGF signalling comprises reducing the bFGF concentration in the medium to below 100 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 ; or below 1 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2. This can be achieved for example by changing the medium, removing the cells from the medium, or through using up the compound over time.

[0126] In one embodiment, the invention relates to a method of differentiating PSCs into Definitive Endoderm (DE), comprising the steps of:

[0127] • seeding PSCs in a bioreactor to initiate a 3D suspension culture under conditions that allow PSC cluster formation;

[0128] • expanding PSCs in culture for at least 2 days, preferably at least 4 days, while performing one of the following:

[0129] (i) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3, preferably wherein the agitation is continuous;

[0130] (ii) treating the PSC clusters with Blebbistatin, preferably wherein the PSC clusters are treated with 5-15 pM Blebbistatin; and / or (iii) modulating bFGF signalling, preferably wherein modulating bFGF signalling comprises adding 100 to 500 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 ; or adding 1 to 100 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2; and

[0131] • inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise dissociation of PSC clusters, preferably wherein at least 90% of expanded PSCs are capable of differentiating into DE.

[0132] Hence, the invention provides a method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and differentiating expanded PSCs into DE, comprising the steps of: a) seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3; c) culturing the PSC clusters for at least 2 days; and d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation of step d) does not comprise dissociation of PSC clusters.

[0133] In one embodiment, the invention relates to a method of differentiating PSCs into Definitive Endoderm (DE), comprising the steps of:

[0134] • seeding PSCs in a bioreactor to initiate a 3D suspension culture under conditions that allow PSC cluster formation;

[0135] • expanding PSCs in culture for at least 2 days, preferably at least 4 days, while providing a source of agitation to the PSC clusters that produces a volumetric power input of at least 0.4 W / m3; and

[0136] • inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise dissociation of PSC clusters, preferably wherein at least 90% of expanded PSCs are capable of differentiating into DE.

[0137] In one embodiment, the invention also relates to a method of differentiating PSC into Definitive Endoderm (DE), the method comprising a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days; c) treating the PSC clusters with Blebbistatin; and d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation; wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters; preferably wherein at least 90% of expanded PSCs are capable of differentiating into DE; optionally wherein the PSC clusters are treated with 5-15 pM Blebbistatin.

[0138] In one embodiment, the invention relates to a method of differentiating PSCs into Definitive Endoderm (DE), comprising the steps of:

[0139] • seeding PSCs in a bioreactor to initiate a 3D suspension culture under conditions that allow PSC cluster formation;

[0140] • expanding PSCs in culture for at least 2 days, preferably at least 4 days, while treating the PSC clusters with Blebbistatin; and

[0141] • inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise dissociation of PSC clusters, preferably wherein at least 90% of expanded PSCs are capable of differentiating into DE; optionally wherein the PSC clusters are treated with 5-15 pM Blebbistatin. In one embodiment, the invention also relates to a method of differentiating PSC into Definitive Endoderm (DE), the method comprising a) seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days in culture medium, wherein bFGF signalling is modulated during culturing; and c) inducing PSC clusters to differentiate to DE by direct cluster transfer differentiation, wherein the method does not comprise a step of dissociation of PSC clusters before differentiation into DE; wherein at least 90% of PSCs are capable of differentiating into DE; preferably wherein modulating bFGF signalling comprises

[0142] (i) adding 100 to 500 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 ; or

[0143] (ii) adding 1 to 100 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2.

[0144] Even more preferably, modulating bFGF signalling comprises

[0145] (i) adding 300 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 ; or

[0146] (ii) adding 50 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2.

[0147] In one embodiment, the invention relates to a method of differentiating PSCs into Definitive Endoderm (DE), comprising the steps of:

[0148] • seeding PSCs in a bioreactor to initiate a 3D suspension culture under conditions that allow PSC cluster formation;

[0149] • expanding PSCs in culture for at least 2 days, preferably at least 4 days, while modulating bFGF signalling; and

[0150] • inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise dissociation of PSC clusters, preferably wherein at least 90% of expanded PSCs are capable of differentiating into DE; optionally wherein modulating bFGF signalling comprises

[0151] (i) adding 100 to 500 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 ; or

[0152] (ii) adding 1 to 100 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2.

[0153] In all embodiments of the method, the respective intervention during the 3D suspension culture of PSC, namely one or more of agitating of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3, treating the culture with Blebbistatin, or modulating bFGF signalling, takes place at least until the time point when differentiation is initiated. Differentiation is usually initiated by adding differentiation factors or changing the medium to a medium comprising differentiation factors.

[0154] In one embodiment, agitating of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3is continuous, but may be interrupted or slowed down during medium changes or other manipulations of the culture.

[0155] In one embodiment, agitating of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3may be interrupted for up to 24 hours. In one embodiment, agitating of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3may be interrupted for up to 1 hour, for up to 2 hours, for up to 5 hours, for up to 10 hours, or for up to 15 hours. In one embodiment, agitating of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3may be interrupted one or more times for up to 24 hours. In one embodiment, agitating of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3may be interrupted one or more times for up to 1 hour, for up to 2 hours, for up to 5 hours, for up to 10 hours, or for up to 15 hours each time, wherein each interruption may be of a different length.

[0156] In one embodiment, agitating of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3is stopped when differentiation of the PSC clusters in initiated.

[0157] In one embodiment, the invention also relates to a method of differentiating PSC into Definitive Endoderm (DE), the method comprising a) seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days; c) quantifying the expression of one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21; wherein DE-differentiation-defective PSC clusters exhibit lower levels of the one or more markers compared to a threshold value; and d) inducing PSC clusters to differentiate to DE by direct cluster transfer differentiation, wherein the method does not comprise a step of dissociation of PSC clusters before differentiation into DE.

[0158] In one embodiment, the invention also relates to a method of differentiating PSC into Definitive Endoderm (DE), the method comprising a) seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days, preferably for at least 3 days, more preferably for at least 4 days; c) quantifying the expression of the markers KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21; wherein DE-differentiation-defective PSC clusters exhibit lower levels of the markers compared to a threshold value; and d) inducing PSC clusters to differentiate to DE by direct cluster transfer differentiation, wherein the method does not comprise a step of dissociation of PSC clusters before differentiation into DE, wherein the threshold value is determined by comparing expression levels of the one or more markers between known DE-differentiation-defective and DE-differentiation-capable PSC clusters.

[0159] In one embodiment, the PSC clusters are cultured continuously without passaging for at least 3 days before differentiation into DE. In one embodiment, the PSC clusters are cultured continuously without passaging for at least 4 days, at least 5 days, at least 6 days, or at least 7 days before differentiation into DE. In a preferred embodiment, the PSC clusters are cultured continuously without passaging for at least 4 days before differentiation into DE.

[0160] In one embodiment, the method of the invention further comprises the steps of growing PSC in a 2D adherent culture, followed by dissociating PSC prior to step a).

[0161] In one embodiment, the method of the invention further comprises harvesting of PSCs after expansion but before differentiation. Harvesting may be followed by cryopreservation of PSCs.

[0162] In one embodiment, the method of the invention further comprises harvesting of DE cells after differentiation. Harvesting may be followed by cryopreservation of DE cells.

[0163] In one embodiment, the PSC clusters have a mean diameter of < 300 pm. In a preferred embodiment, the PSC clusters have a mean diameter of < 200 pm. In a more preferred embodiment, the PSC clusters have a mean diameter of < 150 pm.

[0164] In another embodiment, the PSC clusters have a mean diameter of 50 pm to 300 pm. In a preferred embodiment, the PSC clusters have a mean diameter of 50 pm to 200 pm. In a more preferred embodiment, the PSC clusters have a mean diameter of 50 pm to 150 pm. In an even more preferred embodiment, the PSC clusters have a mean diameter of 70 pm to 140 pm.

[0165] 3D suspension culture

[0166] Methods of PSC 3D suspension culture are known in the art, see for example Manstein et al., 2021 , Stem Cells Transl. Med. 10(7): 1063-1080; Ullmann et al., 2024, Stem Cell Res. Ther. 15(89); or Borys et al., 2024, Stem Cell Res. Ther. 15(232).

[0167] The method is performed in a bioreactor. Particularly, in a preferred embodiment, the bioreactor is a stirred-tank bioreactor. Other bioreactors with controlled volumetric force input are also considered, such as vertical-wheel bioreactors and rocking-motion bioreactors.

[0168] The skilled person is aware of sources of agitation for bioreactors that are suitable for 3D suspension cultures. These include, but are not limited to, rotors; stirring bars, such as magnetic stirring bars; and shakers, such as orbital shakers, wave shakers or rotating shakers.

[0169] In one embodiment, the source of agitation is a rotor, particularly an impeller. An impeller is a driven rotor. An impeller typically has at least 3 blades that are angled between 0 and 90°. Exemplarily, the impeller used in the stirred-tank bioreactor may have 3-8 blades, wherein each blade is at an angle between 40° and 60°.

[0170] Preferably, the bioreactor system comprises pH and / or dissolved oxygen control. In one embodiment, the bioreactor comprises a perfusion system.

[0171] Compared to simple 3D systems such as spinner flasks, using bioreactors (i.e. stirred-tank bioreactors, STRs) during the expansion phase allows to obtain greatly increased expansion rates by controlling certain media parameters (e.g. pH, dissolved oxygen), removal of metabolites with adverse effects (e.g. lactate) and enabling replenishment of nutrients.

[0172] In one embodiment, in step a) 3D suspension culture is initiated by seeding PSC at a cell density of 0.1 x 106cells / ml to 1 x 106cells / ml, preferably 0.4 x 106cells / ml to 0.6 x 106cells / ml, more preferably about 0.5 x 106cells / ml.

[0173] In one embodiment, the PSCs are cultured in PSC growth media. In one embodiment, the medium is serum-free.

[0174] In one embodiment, the PSC growth media comprises one or more components from the list consisting of human albumin, human TGF-pi , basic fibroblast growth factor (bFGF), nonionic surfactant, Vitamin B1 and Rock Inhibitor.

[0175] In one embodiment, the PSC growth media comprises 1-3 ng / ml human TGF-pi , 1-50ng / ml bFGF, 0.1 %-1 % non-ionic surfactant, 10-25 pM Vitamin B1 and 5-15pM Rock Inhibitor.

[0176] In a preferred embodiment, the PSC growth media comprises 2ng / ml human TGF-pi, 50ng / ml bFGF, 0.1% non-ionic surfactant, 18 pM Vitamin B1 and 10pM Rock Inhibitor.

[0177] In one embodiment, the growth factor is selected from and basic fibroblast growth factor (bFGF). The canonical or full-length bFGF is a 154 amino acid protein with the sequence of SEQ ID NO: 2. Truncated version of bFGF comprising the full core bFGF domain and heparin binding domains are commonly used for cell culture, such as a 146 amino acid version, as depicted in SEQ ID NO: 1 , which is truncated by 8 amino acids from the N terminus.

[0178] In one embodiment, the bFGF used in the methods of the invention is a full length bFGF or a truncated version which retains FGFR binding and heparin binding function.

[0179] In one embodiment, the truncated version of bFGF is truncated at the N-terminus, preferably the truncated version of bFGF is truncated at the N-terminus by up to 22 amino acids. In one embodiment, the truncated version of bFGF is truncated at the N-terminus by at least 8 and up to 22 amino acids, preferably, he truncated version of bFGF is truncated at the N-terminus by 8 amino acids

[0180] Hence, in one embodiment, the bFGF is selected from the group consisting of a 146 amino acid bFGF and a 154 amino acid bFGF. In one embodiment, the 146 amino acid bFGF has the amino acid sequence depicted in SEQ ID NO: 1 (PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEER GWSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRT GQYKLGSKTGPGQKAILFLPMSAKS).

[0181] In a preferred embodiment, the basic fibroblast growth factor is recombinant human bFGF 146 amino acid version at a concentration of 100-500ng / ml, preferably 300ng / ml. This is available, for example, from Bio-Techne® with the catalogue number 233-FB.

[0182] Surprisingly, the inventors found that the 154 amino acid bFGF protein was effective at retaining PSC clusters DE-differentiation capability at lower concentrations when added during the expansion phase of the PSC culture than truncated versions (see Example 7, Figure 6). This was not expected, since the N-terminally truncation has been shown to have no effect on growth factor activity (Klagsbrun et al. 1986, PNAS 84:1839-1843; Kastrup et al. 1996, Acta Cryst. D53:160-168).

[0183] Hence, in an even more preferred embodiment, the basic fibroblast growth factor is recombinant human bFGF 154 amino acid version at a concentration of 1 to 100 ng / mL, preferably 50 ng / mL. This is available for example from Peprotech® with the catalogue number 100-18B.

[0184] In one embodiment, the 154 amino acid version of bFGF has the sequence depicted in SEQ ID NO: 2 (AAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKL QLQAEERGVVSI KGVCAN RYLAM KEDGRLLASKCVTDECFFFERLESN NYNTYRSRKYTS WYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS).

[0185] In one embodiment, the non-ionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 80, macrogol 15 hydroxy stearate, and macrogolglycerol ricinoleate. Preferably, the non-ionic surfactant is poloxamer 188.

[0186] In one embodiment, the PSC culture is maintained at pH 7.0 to pH 7.4. Preferably, PSC culture is maintained at pH 7.1. Expansion ofPSCs

[0187] In one embodiment, the PSC is selected from induced PSC (iPSC) and embryonic stem cell (ESC). In a preferred embodiment, the PSC is an iPSC.

[0188] In one embodiment, the PSC is a mammalian PSC, preferably a human iPSC or human ESC.

[0189] In one embodiment, the PSC is a iPSC cell line selected from the group consisting of iPSC6.2 / GibcoEpi, iPS11 , iPS15, F002.1A.13, hiPSC-1 , hiPSC-2, UPSC-GR1.1 , LiPSC- GR1.2, HEL24.3, HEL113.5-corrected, CGT-RCiB-10, MHHi001-A, MHHi006-A, MHHi008-A, MHHi008-B, MHHi008-C, VC645-9, VC913-5, VC618-3, VC646-1 , iPS 1016, iPS 1031 , QHJI01 / -14, RWMH09 / -15 / -23, DRXT18 / -28, RJWI, KTRH05 / -26, D2L01 , and D2L03.

[0190] Preferably, it is a clinical grade or GMP-grade iPSC line. It should be noted that iPSC lines can also be generated in a patient-specific manner for personalized / autologous applications, preferably as GMP-grade / clinical grade lines.

[0191] Similarly, many hESC lines, including also clinical / GMP-grade hESC lines are available for use in context with the invention, such as H1 , H9, HLIES8, HAD-C100 / -102 / -106, CyT49, MEL1 , KCL031 / -33 / -34 / -37 / -38 / -40, KARO1 , KthES11 / -12 / -13 / -14 / -15, RC-09 / -11 / -12 / -13 / - 14 / -15 / -16 / -17, I6, MAN10 / -11 / -12, MAN14 / -15 / -16, ESI-013 / -014 / -017 / -051 / -027 / -035 / -049 / - 053, MasterShef2 / -4 / -10 / -11 , Q-CTS-hESC-1 / -2, Yazd4 / -5 / -6 / -7. Preferably, it is a clinical grade or GMP-grade ESC line.

[0192] In one embodiment, the PSC are expanded by at least 1.5 fold by day 4 of culture compared to the starting material.

[0193] In one embodiment, the PSC are expanded by at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, or at least 10 by day 4 of culture compared to the starting material.

[0194] In one preferred embodiment, the PSC are expanded by at least 8 fold by day 4 of culture compared to the starting material.

[0195] In another embodiment, the PSC are expanded by between 1.5 and 10 fold by day 4 of culture compared to the starting material. In one embodiment, the PSC are expanded by between 1.5 and 10 fold, between 2 and 10 fold, between 2.5 and 10 fold, between 3 and 10 fold, between 3.5 and 10 fold, between 4 and 10 fold, between 4.5 and 10 fold, between 5 and 10 fold, between 5.5 and 10 fold, between 6 and 10 fold, between 6.5 and 10 fold, between 7 and 10 fold, between 7.5 and 10 fold, between 8 and 10 fold, between 8.5 and 10 fold, between 9 and 10 fold, or between 9.5 and 10 fold by day 4 of culture compared to the starting material.

[0196] In a preferred embodiment, the PSC are expanded by between 5 and 10 fold by day 4 of culture compared to the starting material. In a more preferred embodiment, the PSC are expanded by between 8 and 10 fold by day 4 of culture compared to the starting material.

[0197] In another embodiment, the PSC are expanded by at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, or at least 100 by day 4 of culture compared to the starting material.

[0198] In another embodiment, the PSC are expanded by at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90 fold, at least 95 fold, or at least 100 by day 7 of culture compared to the starting material.

[0199] Culture Volume and Stirring Speed

[0200] In one embodiment, the culture volume is between 0.1 L and 2000L. The skilled person is aware that the stirring speed (revolutions per minute, rpm) must be adapted to the volume of the culture.

[0201] In one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm.

[0202] In one embodiment, the volumetric power input is between > 0.25 W / m3to < 2.5 W / m3. Hence, for each culture volume, the rpm must be adapted accordingly.

[0203] Thus, in one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.25 W / m3to < 2.5 W / m3. Thus, in one embodiment, the culture volume is between about 0.1 and about 2000L and the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.25 W / m3to < 2.5 W / m3, wherein the rpm is adapted to produce the required volumetric power input in the given culture volume.

[0204] In one embodiment, the culture volume is between about 0.1 and about 2000L;the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2 W / m3, wherein the rpm is adapted to produce the required volumetric power input in the given culture volume.

[0205] In one embodiment, the culture volume is between about 1L and about 2000L; the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 1 W / m3to < 2 W / m3, wherein the rpm is adapted to produce the required volumetric power input in the given culture volume.

[0206] In one embodiment, the culture volume is between about 1L and about 2000L, between about 1 L and about 1500L, between about 1 L and about 1000L, between about 1 L and about 500L, between about 1L and about 200L, between about 1 L and about 100L, between about 1 L and about 50L, between about 1 L and about 20L, between about 1 L and about 5L, or between about 1L and about 2L; the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2 W / m3, wherein the rpm is adapted to produce the required volumetric power input in the given culture volume.

[0207] In one embodiment, the culture volume is between about 1 L and about 2000L, between about 2L and about 2000L, between about 5L and about 2000L, between about 10L and about 2000L, between about 20L and about 2000L, between about 50L and about 2000L, between about 100L and about 2000L, between about 200L and about 2000L, or between about 500L and about 2000L; the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2 W / m3, wherein the rpm is adapted to produce the required volumetric power input in the given culture volume.

[0208] In a preferred embodiment, the culture volume is between about 0.23L and about 100L; the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2 W / m3, wherein the rpm is adapted to produce the required volumetric power input in the given culture volume.

[0209] In one embodiment, the culture volume is between about 0.23L and about 5L; the stirring speed is between > 35 rpm to <150 rpm and the volumetric power input is between > 0.4 W / m3to < 2 W / m3, wherein the rpm is adapted to produce the required volumetric power input in the given culture volume.

[0210] In one embodiment, the culture volume is about 0.1 L and the stirring speed is between > 80 rpm to <150 rpm.

[0211] In one embodiment, the culture volume is about 0.23 L and the stirring speed is between > 75 rpm to <135 rpm.

[0212] In one embodiment, the culture volume is about 1.5 L and the stirring speed is between > 73 rpm to < 110 rpm.

[0213] In one embodiment, the culture volume is about 10 L and the stirring speed is between > 50 rpm to < 95 rpm.

[0214] In one embodiment, the culture volume is about 50 L and the stirring speed is between > 45 rpm to < 85 rpm.

[0215] In one embodiment, the culture volume is about 100 L and the stirring speed is between > 40 rpm to < 80 rpm.

[0216] In one embodiment, the culture volume is about 500 L and the stirring speed is between > 35 rpm to < 70 rpm.

[0217] In one embodiment, the culture volume is about 1000 L and the stirring speed is between > 30 rpm to < 65 rpm.

[0218] In one embodiment, the culture volume is about 2000 L and the stirring speed is between > 25 rpm to < 60 rpm.

[0219] In one embodiment, the culture volume is about 0.1 L and the stirring speed is between > 80 rpm to <150 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0220] In one embodiment, the culture volume is about 0.23 L and the stirring speed is between > 75 rpm to <135 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range. In one embodiment, the culture volume is about 1.5 L and the stirring speed is between > 64 rpm to < 110 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0221] In one embodiment, the culture volume is about 10 L and the stirring speed is between > 50 rpm to < 95 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0222] In one embodiment, the culture volume is about 50 L and the stirring speed is between > 45 rpm to < 85 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0223] In one embodiment, the culture volume is about 100 L and the stirring speed is between > 40 rpm to < 80 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0224] In one embodiment, the culture volume is about 500 L and the stirring speed is between > 35 rpm to < 70 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0225] In one embodiment, the culture volume is about 1000 L and the stirring speed is between > 30 rpm to < 65 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0226] In one embodiment, the culture volume is about 2000 L and the stirring speed is between > 25 rpm to < 60 rpm, and the differentiation potential into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a stirring speed outside of this range.

[0227] In one embodiment, the invention also relates to a method of expanding PSC capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture, comprising the steps of: a) incubating PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3.

[0228] In another embodiment, the invention also relates to a method of expanding PSC capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture with direct cluster transfer, comprising the steps of: a) incubating PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC clusters; wherein the culture volume is about 0.1 L and the stirring speed is between > 80 rpm to <150 rpm; or wherein the culture volume is about 0.23 L and the stirring speed is between > 75 rpm to <135 rpm, or wherein the culture volume is about 1.5 L and the stirring speed is between > 64 rpm to < 110 rpm, or wherein the culture volume is about 10 L and the stirring speed is between > 50 rpm to < 95 rpm, or wherein the culture volume is about 50 L and the stirring speed is between > 45 rpm to < 85 rpm, or wherein the culture volume is about 100 L and the stirring speed is between > 40 rpm to < 80 rpm, or wherein the culture volume is about 500 L and the stirring speed is between > 35 rpm to < 70 rpm, or wherein the culture volume is about 1000 L and the stirring speed is between > 30 rpm to < 65 rpm, or wherein the culture volume is about 2000 L and the stirring speed is between > 25 rpm to < 60 rpm. Optionally, the method further comprises culturing the PSC clusters for at least 2 days.

[0229] Formula

[0230] The volumetric power input is determined using the formula Equation (I): wherein P = Power [W],

[0231] V = liquid volume [m3],

[0232] N = impeller rotational speed [s’1],

[0233] D = impeller diameter [m], w = impeller width [m], bn= number blades of impeller [-],

[0234] Dv = diameter of bioreactor [m],

[0235] 0 = blade angle [°], p = liquid density [kg / m3], wherein the liquid density is determined for the cell-free fraction of the liquid contained in the bioreactor.

[0236] Cell populations

[0237] In a second aspect, the invention also provides PSCs capable of differentiating into Definitive Endoderm (DE) obtained by the any one of the methods described above. In one embodiment, in order to produce a population of PSCs capable of differentiating into Definitive Endoderm (DE), the final step of the inventive method, step d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation; is omitted, <The method therefore produces a population of undifferentiated PSCs that have a high capacity (over 90%) of differentiating into DE by direct cluster differentiation.

[0238] In one embodiment, at least 90% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 2 days in culture, wherein the PSC clusters are not dissociated before differentiation. In one embodiment, at least 93% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 2 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0239] In one embodiment, at least 95% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 2 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0240] In one embodiment, at least 90% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 3 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0241] In one embodiment, at least 93% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 3 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0242] In one embodiment, at least 95% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 3 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0243] In one embodiment, at least 90% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 4 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0244] In one embodiment, at least 93% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 4 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0245] In one embodiment, at least 95% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after at least 4 days in culture, wherein the PSC clusters are not dissociated before differentiation.

[0246] In some embodiments, PSC clusters are not dissociated and reaggregated before differentiation.

[0247] In a third aspect, the invention also provides a population of Definitive Endoderm cells produced by direct cluster transfer differentiation of the population of PSC described above. The presence of undifferentiated PSCs can be determined using pluripotency markers, such as 0CT4, NANOG, LIN28A, ESRG, S0X2, ALPL, SSEA3, SSEA4, TRA-1-60 and / or TRA-1- 81.

[0248] The presence of DE cells can be determined using endoderm markers such as S0X17, CXCR4, CD117, C-KIT, F0XA2 and EPCAM. Preferably, CXCR4 and EPCAM are used for identification and quantification of DE cells.

[0249] In one embodiment, the presence of DE-differentiation-defective PSCs can be determined using one or more markers selected from KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21 , wherein the expression level of the one or more markers is higher in DE-differentiation-defective PSCs compared to the expression level in DE-differentiation- capable PSCs.

[0250] Hence, in a further aspect, the invention provides a population of DE-differentiation-defective PSCs, wherein the population of DE-differentiation-defective PSCs expresses one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21 , or any combination thereof at higher levels than a population of DE-differentiation-competent PSCs.

[0251] In one embodiment, the population of DE-differentiation-defective PSCs expresses at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21 , or any combination thereof at higher levels than a population of DE-differentiation-competent PSCs.

[0252] In one embodiment, the population of DE-differentiation-defective PSCs expresses one, two, three, four, five, six, seven, or all eight markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, S100A4 and S0X21, or any combination thereof at higher levels than a population of DE-differentiation-competent PSCs.

[0253] In one embodiment, the population of DE-differentiation-defective PSCs expresses all eight markers KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21 at higher levels than a population of DE-differentiation-competent PSCs.

[0254] Likewise, the invention provides a population of DE-differentiation-competent PSCs, wherein the population of DE-differentiation-competent PSCs expresses one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, S100A4 and S0X21, or any combination thereof at lower levels than a population of DE- differentiation-defective PSCs.

[0255] In one embodiment, the population of DE-differentiation-competent PSCs expresses at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21, or any combination thereof at lower levels than a population of DE-differentiation-defective PSCs.

[0256] In one embodiment, the population of DE-differentiation-competent PSCs expresses one, two, three, four, five, six, seven, or all eight markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21, or any combination thereof at lower levels than a population of DE-differentiation-defective PSCs.

[0257] In one embodiment, the population of DE-differentiation-competent PSCs expresses all eight markers KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and S0X21 at lower levels than a population of DE-differentiation-defective PSCs.

[0258] DE-competence is defined as at least 90% of PSCs of a population of PSCs being capable of differentiating into DE. In one embodiment, the population of PSCs comprises at least one cluster of PSCs.

[0259] Hence, in one embodiment, the invention provides a method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) performing one or more of the following steps:

[0260] (i) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3;

[0261] (ii) treating the PSC clusters with Blebbistatin, preferably with 5-15 pM Blebbistatin for at least 24 hours; and / or

[0262] (iii) modulating bFGF signalling; preferably wherein modulating bFGF signalling comprises adding about 300 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1; or adding about 50 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2; and optionally c) culturing PSC clusters for at least 2 days, preferably at least 3 days, more preferably at least 4 days; wherein the PSCs express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, S100A4 and SOX21, or any combination thereof at lower levels than a population of DE-differentiation-defective PSCs.

[0263] Further Embodiments

[0264] The invention is also described by the following items:

[0265] 1. A method of differentiating PSCs into Definitive Endoderm (DE) after 3D suspension culture, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) performing one or more of the following steps:

[0266] (i) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3;

[0267] (ii) treating the PSC clusters with Blebbistatin, preferably with 5-15 pM Blebbistatin for at least 24 hours; and / or

[0268] (iii) modulating bFGF signalling; preferably wherein modulating bFGF signalling comprises adding about 300 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1; or adding about 50 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2; c) culturing PSC clusters for at least 2 days, preferably at least 3 days, more preferably at least 4 days; and d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters; and wherein at least 90% of expanded PSCs are capable of differentiating into DE.

[0269] 2. A method of expanding pluripotent stem cells (PSC) capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) performing one or more of the following steps:

[0270] (i) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3;

[0271] (ii) treating the PSC clusters with Blebbistatin, preferably with 5-15 pM Blebbistatin for at least 24 hours; and / or

[0272] (iii) modulating bFGF signalling; preferably wherein modulating bFGF signalling comprises adding about 300 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1; or adding about 50 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2; and c) culturing PSC clusters for at least 2 days, preferably at least 3 days, more preferably at least 4 days; and optionally d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters; and wherein at least 90% of expanded PSCs are capable of differentiating into DE.

[0273] 3. A method of differentiating PSCs into Definitive Endoderm (DE) after 3D suspension culture comprising the following steps: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC, wherein the culture volume is between about 0.1 and about 2000L and source of agitation provides a stirring speed of between > 35 rpm to <150 rpm; c) inducing PSC clusters to differentiate into DE by direct cluster differentiation; wherein at least 90% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 2 days in culture, preferably after at least 3 days in culture; wherein direct cluster transfer differentiation does not comprise a step of PSC cluster dissociation.

[0274] 4. A method of differentiating PSCs into Definitive Endoderm (DE) after 3D suspension culture comprising the following steps: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC, wherein the culture volume is about 1.5 L and the source of agitation provides a stirring speed of between > 64 rpm to <135 rpm; c) inducing PSC clusters to differentiate into DE by direct cluster differentiation; wherein at least 90% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 2 days in culture, preferably after at least 3 days in culture; wherein direct cluster transfer differentiation does not comprise a step of PSC cluster dissociation.

[0275] 5. A method of differentiating PSCs into Definitive Endoderm (DE) after 3D suspension culture comprising the following steps: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC, wherein the culture volume is between about 10 L and the source of agitation provides a stirring speed of between > 50 rpm to <95 rpm; c) inducing PSC clusters to differentiate into DE by direct cluster differentiation; wherein at least 90% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 2 days in culture, preferably after at least 3 days in culture; wherein direct cluster transfer differentiation does not comprise a step of PSC cluster dissociation.

[0276] 6. A method of differentiating PSCs into Definitive Endoderm (DE) after 3D suspension culture comprising the following steps: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC, wherein the culture volume is about 100L and the source of agitation provides a stirring speed of between > 50 rpm to <95 rpm; c) inducing PSC clusters to differentiate into DE by direct cluster differentiation; wherein at least 90% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 2 days in culture, preferably after at least 3 days in culture; wherein direct cluster transfer differentiation does not comprise a step of PSC cluster dissociation.

[0277] 7. A method of differentiating PSCs into Definitive Endoderm (DE) after 3D suspension culture comprising the following steps: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC, wherein the culture volume is about 1000L and the source of agitation provides a stirring speed of between > 50 rpm to <95 rpm; c) inducing PSC clusters to differentiate into DE by direct cluster differentiation; wherein at least 90% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 2 days in culture, preferably after at least 3 days in culture; wherein direct cluster transfer differentiation does not comprise a step of PSC cluster dissociation.

[0278] 8. A method of differentiating PSCs into Definitive Endoderm (DE) after 3D suspension culture comprising the following steps: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; and b) providing a source of agitation of the PSC clusters that produces a volumetric power input of between > 0.4 W / m3to < 2.5 W / m3; c) inducing PSC clusters to differentiate into DE by direct cluster differentiation; wherein at least 90% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 2 days in culture, preferably after at least 3 days in culture; wherein direct cluster transfer differentiation does not comprise a step of PSC cluster dissociation.

[0279] 9. A method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) performing one or more of the following steps:

[0280] (i) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3;

[0281] (ii) treating the PSC clusters with Blebbistatin, preferably with 5-15 pM Blebbistatin for at least 24 hours; and / or

[0282] (iii) modulating bFGF signalling; preferably wherein modulating bFGF signalling comprises adding about 300 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1; or adding about 50 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2; and optionally c) culturing PSC clusters for at least 2 days, preferably at least 3 days, more preferably at least 4 days.

[0283] 10. A method of detecting DE-differentiation-defective PSC clusters, the method comprising the steps of a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days; and c) quantifying the expression levels of one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21; wherein DE-differentiation-defective PSC clusters exhibit lower expression levels of the one or more markers compared to a threshold value; optionally wherein the threshold value is determined by comparing expression levels of the one or more markers between known DE-differentiation-defective and DE-differentiation- capable PSC clusters.

[0284] 11. Method according to any one of the preceding items, wherein PSCs capable of differentiating into Definitive Endoderm express lower levels of one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21 compared to DE-differentiation-defective iPSC clusters.

[0285] 12. Method according to any one of the preceding items, wherein PSCs capable of differentiating into Definitive Endoderm express lower levels of the markers KDR, CLDN4, CXCL5, DMTN, DYNC2I1 , IGFBP3, S100A4 and SOX21 compared to DE-differentiation- defective iPSC clusters.

[0286] 13. Method according to any one of the preceding items, wherein the source of agitation is a stirrer.

[0287] 14. Method according to any one of the preceding items, wherein the method is performed in a stirred-tank bioreactor.

[0288] 15. Method according to any one of the preceding items, at least 93% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 3 days in culture. 16. Method according to any one of the preceding items, at least 95% of PSCs are capable of differentiating into Definitive Endoderm (DE) by direct cluster transfer differentiation after at least 3 days in culture.

[0289] 17. The method according to any one of the preceding items, wherein the PSC do not become DE differentiation-defective after at least 3 days in culture.

[0290] 18. The method according to any one of the preceding items, wherein the PSC are cultured for at least 3, at least 4, at least 5, at least 6, or at least 7 days before differentiation into DE.

[0291] 19. The method according to any one of the preceding claims, wherein the PSC clusters have a mean diameter of 50 pm to 300 pm.

[0292] 20. A DE-differentiation-competent iPSC cluster expressing lower levels of one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21 compared to DE-differentiation-defective iPSC clusters.

[0293] 21. A DE-differentiation-defective iPSC cluster expressing higher levels of one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21 compared to DE-differentiation-competent iPSC clusters.

[0294] Examples

[0295] Example 1 : 3D Suspension Culture

[0296] Experimental work on cell expansion and differentiation used human iPSC line UPSC-GR1.1 (Baghbaderani et al., Stem Cell Reports. 2015 Oct 13;5(4):647-59).

[0297] To initiate the bioreactor process, 2D expanded (i)PSCs are dissociated with Accutase (enzymatic solution), transferred into (i)PSC growth media (iPS-Brew GMP Medium (Miltenyi Biotec) supplemented with 50 ng / mL bFGF (either 146 amino acid variant according to SEQ ID NO: 1 or 154 amino acid variant according to SEQ ID NO: 2), 0.1% Poloxamer 188, 18.163 pM Vitamin B1, 10pM Rock Inhibitor), counted and seeded at a cell density of 0.5 x 106cells / mL in the respective stirred tank bioreactor system.

[0298] The 3D suspension culture protocol is an adapted version of Manstein et al. STAR Protoc. 2021 Dec 9;2(4): 100988. (PubMed ID: 34917976).

[0299] The bioreactor process is run with a triggered pH control at 7.1 , which means that the pH naturally drops to the intended control value (here 7.1) and once this value is reached the control starts. The pH is maintained by reducing the CO2 concentration in the gas stream from initially 5% and the addition of 1M NaHCCh. In addition, the dissolved oxygen levels (DO, 100% calibrated at air saturation) are controlled in a triggered manner at 40%. The levels are maintained by an increase of the oxygen ratio in the gas stream. After 24 hours of culture, a constant perfusion is initiated. The perfusion rate is set to 1 reactor volume per day for the first 24 h of perfusion, afterwards it is increased to 1.5 reactor volumes for an additional 24 h, before it is increased to 3 reactor volumes per day.

[0300] Example 2: Bioreactor Systems

[0301] For this experiment three different bioreactor systems were used.

[0302] The DASGIP® Bioblock bioreactor system is a flat bottom stirred tank bioreactor (Eppendorf, vessel diameter of 0.1 m, impeller diameter of 0.053 m, impeller width of 0.0155 m, number of blades of 8, blade angle of 60°) that was used with a culture volume of 500 mL at stirring speeds of 45, 60 and 70 rpm.

[0303] The Applikon MiniBio 500 bioreactor system is a hemispherical bottom stirred tank bioreactor (Getinge, vessel diameter of 0.0705 m, impeller diameter of 0.038 m, impeller width of 0.03 m, number of blades of 3, blade angle of 45°) that was used with a culture volume of 230 mL at stirring speeds of 67, 75, 82, 89 and 112 rpm.

[0304] The Applikon Bio 3L bioreactor system is a hemispherical bottom stirred tank bioreactor (Getinge, vessel diameter of 0.1245 m, impeller diameter of 0.06 m, impeller width of 0.055 m, number of blades of 3, blade angle of 45°) that was used with a culture volume of 1500 mL at stirring speeds of 55, 73 and 92 rpm.

[0305] Example 3: Volumetric power input calculation

[0306] In addition to stirring speed (rpm) and culture volume (L), volumetric power input for each bioreactor system was calculated for each of the different stirring speeds.

[0307] Based on the previously mentioned parameters and calculation based on equation (I), this results to the volumetric power input values depicted in Table 1.

[0308] Equation (I):

[0309] P = Power [W]

[0310] V = liquid volume [m3] N = impeller rotational speed [s-1]

[0311] D = impeller diameter [m] w = impeller width [m] bn= number blades of impeller [-]

[0312] Dv = diameter of bioreactor [m] 0 = blade angle [°] p = liquid density [kg / m3], wherein the liquid density is determined for the cell-free fraction of the liquid contained in the bioreactor.

[0313]

[0314] Example 4: Differentiation into Definitive Endoderm

[0315] After expanding the iPSCs in the previously described settings for two, three or four days, iPSC clusters were transferred into 6 well plates to initiate differentiation towards definitive endoderm (DE). In order to achieve comparability between the approaches, the cell density was adjusted to 1 x 106cells / mL in the six well plates before differentiation initiation.

[0316] The basal medium that is used for all three days of differentiation (called subsequently “BMI ”) consists of MCDB-131 supplemented with 2.5 g / L NaHCO3, 7.5 mM Glucose, 2mM GlutaMAX, 1 g / L recombinant human serum albumin, 0.02 % (v / v) Insulin-Transferrin- Selenium-Ethanolamine (ITS -X) and 1.5% (v / v) Penicillin-Streptomycin.

[0317] To initiate the differentiation, the cell density of the bioreactor culture is determined. Afterwards, a volume containing an amount of intact, non-dissociated clusters corresponding to a total of 5.5 x 106cells is transferred from the bioreactor into a 15 mL conical tube, and clusters are allowed to settle. Subsequently, the supernatant is removed, the cell pellet is washed with 5.5 mL MCDB-131 , clusters are allowed to settle again, the supernatant is removed and clusters are resuspended in 5.5 mL BMI + 10 pM of the ROCK Inhibitor Y- 27632 + 100 ng / mL Activin A + 5 ng / mL BMP4 + 5ng / mL FGF-2 + 10 ng / mL Heparin + 1 pM CHIR99021 + 0.5 mM Ascorbic Acid, to obtain a starting cell concentration of 1.0 x 106cells / mL. This cell suspension is transferred into Clear Flat Bottom Ultra-Low Attachment 6- Well Plates (Corning, 3471). The plate is transferred to an orbital shaking platform with a shaking throw of 25 mm and cultured at 100 rpm in an incubator at 37°C and 5% CO2.

[0318] 24 h later the cell suspension is transferred from the 6 well plate into a 15 mL conical tube, and clusters are allowed to settle. Subsequently, the supernatant is removed, the cell pellet is washed with 5.5 mL MCDB-131 , clusters are allowed to settle again, the supernatant is removed and clusters are resuspended in 5.5 mL BMI + 100 ng / mL Activin A + 5ng / mL FGF- 2 + 10 ng / mL Heparin + 0.5 mM Ascorbic Acid. Afterwards, the cell suspension is transferred back into the 6-well plate and cultured in the orbital shaking platform as described before.

[0319] Another 24 h (= 48 h after differentiation initiation) later the cell suspension is transferred from the 6 well plate into a 15 mL conical tube, and clusters are allowed to settle. Subsequently, the supernatant is removed and clusters are resuspended in 5.5 mL BMI + 100 ng / mL Activin A + 5ng / mL FGF-2 + 10 ng / mL Heparin + 0.5 mM Ascorbic Acid. Afterwards, the cell suspension is transferred back into the 6-well plate and cultured in the orbital shaking platform as described before. Another 24 h (= 72 h after differentiation initiation) later the clusters are dissociated, cells are counted, stained for CXCR4 and EPCAM, and analysed via flow cytometry.

[0320] Figure 1 shows an overview of the experimental set up. Each culture is stirred at three different speeds and on day 2, day 3 and day 4, clusters are differentiated by direct cluster transfer differentiation.

[0321] Figure 2 shows flow cytometry assessment of EPCAM / CXCR4 of 2, 3 and 4 day expanded iPSC clusters in the DASGIP® Bioblock bioreactor system at 500 mL culture volume and given stirring speeds with a 8-blade impeller. After 4 days in culture, the DE differentiation capacity declined to about 62% at slow stirring speed. At a stirring speed of 60rpm and volumetric force input of about 1.186 W / m3in a 500ml culture, DE differentiation was above 90% even after 4 days in culture.

[0322] Figure 3A shows flow cytometry assessment of EPCAM / CXCR4) of 2, 3 and 4 day expanded iPSC clusters in the Applikon MiniBio 500 bioreactor system at 230 mL culture volume and given stirring speeds with a 3-blade impeller. After 4 days in culture, the DE differentiation capacity declined to about 24% at slow stirring speed. At a stirring speed of 112rpm and volumetric force input of about 1.334 W / m3in a 230ml culture, DE differentiation was above 90% even after 4 days in culture.

[0323] Figure 3B shows flow cytometry assessment of DE differentiation (quantified vie flow cytometric analysis of EPCAM / CXCR4) of 2 and 3 day expanded iPSC clusters in the Applikon MiniBio 500 bioreactor system at 230 mL culture volume and with indicated stirring speeds with a 3-blade impeller. Subsequent differentiation was conducted in 6 well plates. After 3 days in culture, DE differentiation capacity declined at slow stirring speed.

[0324] Figure 4 shows flow cytometry assessment of EPCAM / CXCR4 of 2, 3 and 4 day expanded iPSC clusters in the Applikon Bio 3L bioreactor system at 1500 mL culture volume and given stirring speeds with a 3-blade impeller. Subsequent differentiation was conducted in 6 well plates. After 4 days in culture, the DE differentiation capacity declined to about 50% at slow stirring speed. At a stirring speed of 92rpm and volumetric force input of about 1.342 W / m3in a 1500ml culture, DE differentiation was above 90% even after 4 days in culture.

[0325] Example 5: PSC cluster size

[0326] Cluster size in pm was measured for each culturing condition. Results are shown in Table 2. Clusters that led to good DE differentiation results were between about 70pm to about 140- 185pm in diameter, which is scale and vessel configuration dependent.

[0327] Example 6: Blebbistatin treatment reverts DE defective phenotype of PSCs

[0328] The expansion was conducted as previously described at 75 rpm in the Applikon MiniBio 500 bioreactor system at 230 mL culture volume. After three days, clusters were transferred to six well plates where they were further cultured in pluripotency supporting media and either treated with 10 M Blebbistatin or 0.09% DMSO (solvent control) for 24 h. Afterwards these clusters were transferred to differentiation towards DE as previously described. As additional control, clusters that were cultured for four days in bioreactors were also transferred to differentiation.

[0329] Figure 5 shows that treatment of cells with 10 M Blebbistatin for 24 h is able to revert the DE defective phenotype in PSCs so that these cells are able to differentiate efficiently into DE. In addition, Figure 5 shows that culture for 24 h in a 6 well plate does not impact cell differentiation potential towards DE compared to cells that were maintained in bioreactors.

[0330] Example 7: bFGF type and concentration influence DE differentiation capacity

[0331] The expansion was conducted as previously described at 75 rpm in the Applikon MiniBio 500 bioreactor system at 230 mL culture volume, with the differences that either the applied bFGF (146 amino acids, SEQ ID NO: 1 vs 154 amino acids, SEQ ID NO: 2) was exchanged, or that the concentration of the 146 amino acid version was increased 6-fold.

[0332] Figure 6 shows that that both modifications to the expansion protocol were able to prevent the DE defectiveness after direct cluster transfer.

[0333] Example 8: Characterization of DE defective cells and molecular marker identification

[0334] Samples of 3D expanded iPSC clusters shown to be either DE differentiation defective or competent were used to conduct Quantitative PCR (qPCR) for potential DE defectiveness target genes.

[0335] Figures 7A and 7B show that DE defective iPSC clusters upregulate at least 8 target genes >1.5-fold compared to DE competent iPSC clusters.

Claims

Claims1. A method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and differentiating expanded PSCs into DE, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3; c) culturing PSC clusters for at least 2 days; and d) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters.

2. The method according to claim 1, wherein the PSC are expanded by at least 1.5 fold by day 3 of culture compared to the starting material.

3. The method according to any one of the preceding claims, wherein the differentiation capacity into Definitive Endoderm (DE) is improved compared to PSC in a 3D suspension culture experiencing a volumetric power input of less than 0.4 W / m3, preferably wherein at least 90% of PSC are capable of differentiating into DE.

4. The method according to any one of the preceding claims, wherein the culture volume is between 0.1L and 2000L and the stirring speed is between > 35 rpm to <150 rpm and optionally the volumetric power input is between > 0.4 W / m3to < 2.5 W / m3.

5. The method according to any one of the preceding claims, wherein the culture volume is about 0.1 L and the stirring speed is between > 80 rpm to <150 rpm; or wherein the culture volume is about 0.23 L and the stirring speed is between > 75 rpm to <135 rpm, or wherein the culture volume is about 1.5 L and the stirring speed is between > 64 rpm to < 110 rpm, or wherein the culture volume is about 10 L and the stirring speed is between > 50 rpm to < 95 rpm, orwherein the culture volume is about 50 L and the stirring speed is between > 45 rpm to < 85 rpm, or wherein the culture volume is about 100 L and the stirring speed is between > 40 rpm to < 80 rpm, or wherein the culture volume is about 500 L and the stirring speed is between > 35 rpm to < 70 rpm, or wherein the culture volume is about 1000 L and the stirring speed is between > 30 rpm to < 65 rpm, or wherein the culture volume is about 2000 L and the stirring speed is between > 25 rpm to < 60 rpm.

6. The method according to any one of the preceding claims, wherein the bioreactor is a stirred-tank bioreactor.

7. The method according to any one of the preceding claims, wherein the PSC are human iPSC or human ESC.

8. The method according to any one of the preceding claims, wherein the PSC do not become DE differentiation-defective after more than 3 days in culture.

9. The method according to any one of the preceding claims, wherein the PSC are cultured for at least 3, at least 4, at least 5, at least 6, or at least 7 days before differentiation into DE.

10. The method according to any one of the preceding claims, wherein the PSC clusters have a mean diameter of 50 pm to 300 pm.

11. A method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and differentiating expanded PSCs into DE, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) treating the PSC clusters with Blebbistatin; c) culturing PSC clusters for at least 2 days; andd) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters; wherein at least 90% of expanded PSCs are capable of differentiating into DE; preferably wherein the PSC clusters are treated with 5-15 pM Blebbistatin.

12. A method of expanding PSCs capable of differentiating into Definitive Endoderm (DE) in a 3D suspension culture and differentiating expanded PSCs into DE, comprising the steps of: a) seeding PSC in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) culturing PSC clusters for at least 2 days in culture medium, wherein bFGF signalling is modulated during culturing; and c) inducing PSC clusters to differentiate into DE by direct cluster transfer differentiation, wherein the direct cluster transfer differentiation does not comprise a step of dissociation of PSC clusters; and wherein at least 90% of PSCs are capable of differentiating into DE; preferably wherein modulating bFGF signalling comprises(i) adding 100 to 500 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1 ; or(ii) adding 1 to 100 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2.

13. A population of PSCs capable of differentiating into Definitive Endoderm (DE) obtained by a) seeding PSCs in a bioreactor to initiate 3D suspension culture under conditions that allow PSC cluster formation; b) performing one or more of the following steps:(i) providing a source of agitation of the PSC clusters that produces a volumetric power input of at least 0.4 W / m3;(ii) treating the PSC clusters with Blebbistatin, preferably with 5-15 pM Blebbistatin for at least 24 hours; and / or(iii) modulating bFGF signalling; preferably wherein modulating bFGF signalling comprises adding 100 to 500 ng / mL of a 146 amino acid variant of bFGF according to SEQ ID NO: 1; or adding 1 to 100 ng / mL of a 154 amino acid variant of bFGF according to SEQ ID NO: 2; c) culturing PSC clusters for at least 2 days, preferably at least 3 days, more preferably at least 4 days.

14. The population of PSCs according to claim 13, wherein at least 90% of cells of the population of PSCs are capable of differentiating into DE by direct cluster transfer differentiation after 4 days in culture, wherein the PSC clusters are not dissociated before differentiation.

15. A population of Definitive Endoderm cells produced by direct cluster transfer differentiation of the population of PSC according to any one of claims 13 or 14.

16. A method of detecting DE-differentiation-defective PSC clusters, comprising quantifying the expression levels of one or more markers selected from the group consisting of KDR, CLDN4, CXCL5, DMTN, DYNC2I1, IGFBP3, S100A4 and SOX21; and optionally comparing expression levels of the one or more markers between known DE- differentiation-defective and DE-differentiation-capable PSC clusters to determine a threshold value of the expression level, wherein DE-differentiation-defective PSC clusters exhibit lower expression levels of the one or more markers compared to the threshold value.

17. A population of Definitive Endoderm (DE)-differentiation-defective PSC clusters obtained by the method according to claim 16.

18. The method according to any one of claims 1 to 12 or 16, the population of PSCs of any one of claims 13, 14 or 17, or the population of DE cells according to claim 15, wherein the differentiation into DE is confirmed by determining the expression of one or more DE markers selected from the group consisting of (i) SOX17 alone, (ii) CXCR4 and EPCAM, (iii) CXCR4 and C-KIT, and (iv) FOXA2 alone.

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