Enrichment or isolation of cell clusters

Abstract

Methods of enriching or isolating cell clusters comprising ex vivo differentiated cells, the methods comprising: subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and isolating the cell clusters from the first volume, thereby enriching or isolating the cell clusters. A pharmaceutical composition comprising the enriched or isolated cell clusters. The enriched or isolated cell clusters for use as a medicament. A kit for enriching or isolating cell clusters comprising ex vivo differentiated cells.

Description

[0001] Enrichment or isolation of cell clusters

[0002] The present invention relates to the enrichment or isolation of cell clusters comprising ex vivo differentiated cells, preferably differentiated human stem cells, by density gradient separation. Methods of enrichment or isolation and uses of the enriched or isolated cells are disclosed herein.

[0003] Background

[0004] Pluripotent cells, such as embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and progenitor cells, are promising sources for cell therapies for various diseases and defects. They can be differentiated ex vivo into various types of mature specialized target cells and can be produced in clinically relevant quantities. Multiple of these differentiated cells may be combined, after which they can organize into clusters of cells that show some degree of organotypic functionality.

[0005] However, suboptimal differentiation remains a problem as this results in heterogeneous cell populations comprising non-target cells with undesired characteristics, for example insufficiently differentiated cells or undifferentiated cells, which have insufficient functional or clinical efficacy, or may be tumorigenic. Hence, heterogeneous cell populations increase the volume of cells required for therapeutic purposes, while also presenting additional safety risks.

[0006] Methods for isolating cells from or enriching heterogeneous differentiated cell populations derived from pluripotent cells for target cells typically involve antibody-based strategies such as fluorescence activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). However, these methods disturb the adhesion between cells and put the isolated or enriched cell product not only at risk of cellular stress and cell death, but also at risk of infection due to the high amount of manipulations required. Furthermore, FACS and MACS are generally not suitable for isolation or enrichment of (large) cell clusters as they rely on the dissociation of cells into single cells. Hence, FACS and MACS present various difficulties for large-scale manufacturing of therapeutic cell products.

[0007] Thus, there is a need for methods of enriching or isolating target cells, in particular target cells comprised in cell clusters, which lowers the total amount of cells used for clinical purposes (transplant volume), improves efficiency of the method, does not disturb connections between cells, and improves safety for clinical purposes by limiting unwanted, non-target cells with potential adverse effects.

[0008] Brief description of the invention

[0009] The present invention discloses a method of enriching or isolating cell clusters comprising ex vivo differentiated cells by using density gradient separation. Purifying cell clusters derived from pluripotent (stem) cells by density gradient separation reduces and / or depletes the cell preparation from non-target cells (e.g. cells not (sufficiently) differentiated or not present in clusters) without disturbing the cytoarchitecture and interconnections of the cells in the cell clusters. The method of the invention is performed without using any antibodies or enzymes, which further helps in reducing any interaction with the cell membrane and connections between the cells thus minimally altering cell signaling and minimally inducing cell stress. The reduced amount of non-target cells in the enriched or isolated cell population results in a lower transplant volume, which will reduce procedure-related risks to patients and may facilitate the use of cell delivery material (e.g. biomaterials).

[0010] In density gradient separation, cell clusters are separated on the basis of their density. Cell cluster size only influences the rate at which cell clusters move until their density is the same as the surrounding density gradient medium. The method of the invention is biocompatible and has no noticeable effect on the biological activity or survival of the cells. Therefore, it is suitable for use in the production of a medicinal cell product. Moreover, the cost of the method is relatively low, it has a low risk of contamination, and it can be easily applied in the manufacturing process of stem cell derived products. The method of the invention is easily scalable, highly reproducible, and clinically applicable for effective purification of pluripotent (stem) cell-derived cell preparations to enrich for or isolate target cells that are most suited for clinical application. Hence, the method of the invention is particularly suitable for obtaining enriched or isolated cell clusters for use in (cell) therapy.

[0011] The object of present invention is therefore to provide a method of enriching cell clusters comprising ex vivo differentiated cells, the method comprising: a. subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and b. isolating the cell clusters from the first volume, thereby enriching the cell clusters.

[0012] The object of present invention is also to provide a method of isolating cell clusters comprising ex vivo differentiated cells, the method comprising: a. subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and b. isolating the cell clusters from the first volume.

[0013] Suitably, the first volume may have a density of from about 1 .0 g / ml to about 1 .3 g / ml.

[0014] Suitably, the density gradient medium or the first volume may have a continuous, linear, discontinuous, segmented, or isokinetic density gradient.

[0015] Suitably, the first volume may have a gradient of densities that is within the range of from about 1 .0 g / ml to about 1 .3 g / ml.

[0016] Suitably, the method may further comprise a third volume having a different density from the first and second volumes. Suitably, one of the second or third volumes may have a higher density than the first volume, and the other of the second or third volume may have a lower density than the first volume.

[0017] Suitably, the density gradient medium or the first volume may comprise a cell gradient medium, a cell maintenance medium, a cell culture medium, a tissue preservation medium, and / or a colloid.

[0018] Suitably, the density gradient medium or the first volume may be about isosmotic.

[0019] Suitably, the density gradient medium or the first volume may have on osmolarity of from about 260 mOsm to about 330 mOsm.

[0020] Suitably, subjecting the suspension may be performed at from 0 °C to about 4 °C, 8 °C, or 12 °C.

[0021] Suitably, the ex vivo differentiated cells may comprise cells derived from stem cells

[0022] Suitably, the ex vivo differentiated cells may comprise cells derived from totipotent stem cells, pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), multipotent stem cells, oligopotent stem cells, embryonic stem cells (ESC), mesenchymal stem cells, epithelial stem cells, or organoids.

[0023] Suitably, the ex vivo differentiated cells may comprise cells derived from mammalian cells, preferably human cells.

[0024] Suitably, the ex vivo differentiated cells may comprise endocrine cells.

[0025] Suitably, the ex vivo differentiated cells may comprise thyroid follicular cells, thyroid parafollicular cells, parathyroid chief cells, parathyroid oxyphil cells, or enteroendocrine cells.

[0026] Suitably, the ex vivo differentiated cells may comprise pancreatic enteroendocrine cells or pancreatic islet cells.

[0027] Suitably, the ex vivo differentiated cells may comprise alpha cells, beta cells, gamma cells (also known as pancreatic polypeptide cells), delta cells and / or epsilon cells.

[0028] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may substantially consist of ex vivo differentiated cells.

[0029] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may substantially consist of ex vivo differentiated cells derived from stem cells.

[0030] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may substantially consist of ex vivo differentiated cells derived from totipotent stem cells, pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), multipotent stem cells, oligopotent stem cells, embryonic stem cells (ESC), mesenchymal stem cells, epithelial stem cells, or organoids.

[0031] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may be organotypic cell clusters.

[0032] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may be organotypic endocrine tissue.

[0033] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may be organotypic pancreatic islets, organotypic thyroid follicles, or organotypic parathyroid follicles.

[0034] Suitably, the method according to the invention may further comprise obtaining the suspension, preferably comprising 3D culturing the cell clusters. Suitably, obtaining the suspension may be enzyme-free.

[0035] Suitably, the method according to the invention may be antibody-free and / or enzyme-free.

[0036] Suitably, the ex vivo differentiated cells may comprise organoids, mesenchymal cells, endothelial cells, or epithelial cells.

[0037] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may be substantially spherical or substantially spheroidal.

[0038] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may have a mean diameter of at least about 20 pm or of from about 20 pm to about 6 mm.

[0039] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may each have at least about 20 cells or each have of from about 20 to about 10000 cells.

[0040] Suitably, the enriched or isolated cell clusters move until their density is the same as the surrounding density gradient medium, wherein the density gradient medium may have a density of from about 1 .0 g / ml to about 1 .3 g / ml.

[0041] Suitably, the enriched or isolated cell clusters may not comprise exocrine cells.

[0042] Suitably, the enriched or isolated cell clusters may each comprise at least 40% or of from 40% to 95% C-peptide or insulin secreting cells of the total cell number of the respective cell cluster.

[0043] Suitably, the enriched or isolated cell clusters may each comprise at least 2% or of from 5% to 25% glucagon secreting cells of the total cell number of the respective cell cluster.

[0044] Suitably, the enriched or isolated cell clusters may each comprise at least 70% or of from 70% to 99% thyroid hormone secreting cells of the total cell number of the respective cell cluster.

[0045] Suitably, the enriched or isolated cell clusters may each comprise at least 5% or of from 5% to 25% calcitonin secreting cells of the total cell number of the respective cell cluster.

[0046] Suitably, the enriched or isolated cell clusters may comprise at least 40% or of from 40% to 95% C-peptide or insulin secreting cells.

[0047] Suitably, the enriched or isolated cell clusters may comprise at least 2% or of from 5% to 25% glucagon secreting cells.

[0048] Suitably, the enriched or isolated cell clusters may comprise at least 70% or of from 70% to 99% thyroid hormone secreting cells.

[0049] Suitably, the enriched or isolated cell clusters may comprise at least 5% or of from 5% to 25% calcitonin secreting cells.

[0050] Suitably, the enriched or isolated cell clusters may have a purity of at least 50%.

[0051] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may comprise target cells.

[0052] Suitably, the enriched or isolated cell clusters may comprise at least 50% target cells of the total number of enriched or isolated cells.

[0053] Suitably, the enriched or isolated cell clusters may comprise at least 50% dithizone positive cells of the total number of enriched or isolated cells. Suitably, the enriched or isolated cell clusters may have a glucose stimulation index of at least

[0054] 1.0.

[0055] Suitably, the enriched or isolated cell clusters may comprise at least 95% viable cells, preferably as determined by fluorescein diacetate (FDA) and propidium iodide (PI) staining.

[0056] Suitably, the enriched or isolated cell clusters may be suitable for cell therapy or suitable for use in cell therapy.

[0057] In one aspect the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the enriched or isolated cell clusters obtained by the method according to the invention.

[0058] In another aspect the invention provides enriched or isolated cell clusters obtained by the method according to the invention, or the pharmaceutical composition according to the invention, for use as a medicament.

[0059] In another aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency, comprising obtaining enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, and administering to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition.

[0060] In another aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency, comprising administering enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition.

[0061] In another aspect the invention provides enriched or isolated cell clusters obtained by the method according to the invention, or the pharmaceutical composition according to the invention, for use in the treatment of a defect, disorder, disease, or deficiency of endocrine cells.

[0062] In one aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency of endocrine cells, comprising obtaining enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, and administering to the subject a therapeutically effective amount of the isolated cell clusters or the pharmaceutical composition.

[0063] In one aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency of endocrine cells, comprising administering enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition.

[0064] Suitably, the defect, disorder, disease, or deficiency comprises type 1 diabetes or hypothyroidism. In another aspect the invention provides a kit for enriching or isolating cell clusters comprising ex vivo differentiated cells comprising: a. a first volume having a density in the range of from about 1 .0 g / ml to about 1 .3 g / ml; b. optionally, a second volume having a higher density compared to the highest density of the first volume; c. optionally, a third volume having a lower density compared to the lowest density of the first volume; and d. optionally, instructions for carrying out the method according to the invention.

[0065] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0066] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0067] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0068] The patent, scientific, and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published, and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail. Various aspects of the invention are described in further detail below.

[0069] Brief description of the figures

[0070] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0071] Figure 1 shows that SC-islets present an organotypic cytoarchitecture and exhibit glucose- stimulated C-peptide secretion. (A) Morphology of the cell clusters at stage 0 (left; scale bar, 100 pm), and stage 7 (right; scale bar Scale bar, 200 pm). SC-islet clusters stained red by dithizone stain. (B) Frequency of C-peptide (C-pep), glucagon (GCG) positive and bihormonal (C-pep / GCG) cells in S7 SC-islets derived from HUES8 (n=12) or RC9 (n=6), as assessed by flowcytometry. (C) Immunostaining for C-peptide (green), and Glucagon, NKX6.1 , PDX1 , MAFA, or NEUROD1 (all in pink) of S7 SC-islets. Scale bars, 50 pm. (D) Electron microscopy images of a donor islet p cell (left) and of a p cell from S7 SC-islets (right) labeled with immunogold staining for insulin. Scale bars, 1 pm. (E) C-peptide content (ng) normalized to DNA content (pg) of S7 SC-islet cells derived from HUES8 (HUES8-islet, n=8) and RC9 (RC9-islet, n=9) or donor islets (n=10). One-way ANOVA; Significance versus donor islet, when indicated. (F) Glucose stimulated C-peptide secretion of donor islets (n=10) and S7 SC-islets derived from HUES8 (HUES8-islet, n=8) or RC9 (RC9-islet, n=9), normalized to the C-peptide content. Cell clusters were incubated at low glucose (1 .67 mM), and subsequently to high glucose (20 mM) and to low glucose (1 .67 mM glucose) again. Two-way ANOVA with Dunnett’s multiple comparison tests; Significance versus basal secretion in low glucose, when indicated. (G) Stimulation index (fold change of C-peptide secretion in high glucose over first low glucose) of donor islets (n=10) and S7 SC-islets derived from HUES8 (HUES8-islet, n=8) or RC9 (RC9-islet, n=9); One-way ANOVA; Significance versus donor islets, when indicated. (H) Oxygen Consumption Rate (OCR) of donor islets (n=4) and S7 SC-islets derived from HUES8 (HUES8-islet, n=2) or RC9 (RC9-islet, n=5) in response to 20 mM glucose (Glucose), oligomycin (Oligo) (5 pM), FCCP (4 pM) and rotenone / antimycinA (AntiA / Rot.) (1 pM / 1 pM). Data is normalized to the basal respiration and presented as percentage. (I) Mitochondrial properties indicating Oxygen Consumption Rate (OCR) normalized to protein content of donor islets (n=4) and S7 SC-islets derived from HUES8 (HUES8-islet, n=2) or RC9 (RC9-islet, n=5). Two-way ANOVA, uncorrected Fishers’s LSD. (J) Frequency of C-peptide (C-pep), glucagon (GCG) positive, bihormonal (C- pep / GCG) and (C-pep / NKX6.1) cells in S7 SC-islets derived from HUES8 (n=16) or RC9 (n=7), as assessed by flowcytometry. (K) C-peptide content (ng) normalized to DNA content (pg) of S7 SC- islet cells derived from HUES8 (HUES8-islet, n=10) and RC9 (RC9-islet, n=9) or donor islets (n=10). One-way ANOVA; Significance versus donor islet, when indicated. Data are presented as a box plot, and the median is shown. (L) Glucose stimulated C-peptide secretion of donor islets (n=10) and S7 SC-islets derived from HUES8 (HUES8-islet, n=10) or RC9 (RC9-islet, n=9), normalized to the C- peptide content. Cell clusters were incubated at low glucose (1 .67 mM), and subsequently to high glucose (20 mM) and to low glucose (1.67 mM glucose) again. Two-way ANOVA with Dunnett’s multiple comparison tests; Significance versus basal secretion in low glucose, when indicated. (M) Stimulation index (fold change of C-peptide secretion in high glucose over first low glucose) of donor islets (n=10) and S7 SC-islets derived from HUES8 (HUES8-islet, n=10) or RC9 (RC9-islet, n=9); One-way ANOVA; Significance versus donor islets, when indicated. All data in Figure 1 are presented as mean ±S.E.M. * P < 0.05, “ P < 0.01 , *** P < 0.001 .

[0072] Figure 2 shows single-cell transcriptomic profiling of SC-p cells. (A) Heatmap showing the average expression of selected genes associated with p cell identity (38) in p cells from donor islets (donor-p, n=3) and S7 SC-p cells derived from HUES8 (HUES8-P, n=2) or RC9 (RC9-P, n=3). (B) Heatmap showing the average expression of genes related to p cell maturation, metabolic sensing and signaling, function and secretion, exocytosis, non-canonical factors and disallowed genes (adapted from (7, 10)) in p cells from donor islets (donor-p, n=3) and S7 SC-p cells derived from HUES8 (HUES8-P, n=2) or RC9 (RC9-P, n=3). (C to E) Violin plot representing the expression of selected genes (from panel B) related to glycolysis and TCA cycle (C), oxidative phosphorylation (D), and insulin secretion (E) in p cells from donor islets (donor-p, n=3) and S7 SC-p cells derived from HUES8 (HUES8-P, n=2) or RC9 (RC9-P, n=3).

[0073] Figure 3 shows engrafted S7 SC-islets in immunodeficient mice. (A) Schematic representation of the in vivo experimental set-up indicating the time points at which glucose challenges were performed. (B) Immunostaining for C-peptide (green), and Glucagon (pink) of S7 SC-islets at 100 days post-engraftment. Scale bar, 20 pM. (C and D) Maximum stimulated human C- peptide concentration in mice engrafted with S7 SC-islets derived from HUES8 (C; day 14 (n=19), day 28 (n=19), day 60 (n=10), day 90 (n=9)) and RC9 (D; day 14 (n=33), day 28 (n=33), day 60 (n=29), day 90 (n=14)); Mixed effect analysis using Dunnett’s multiple comparison; Significance over day 14, when indicated. (E and F) Maximum stimulated human C-peptide concentration in mice engrafted with S7 SC-islets derived from HUES8 (E; day 14 (n=14), day 28 (n=14), day 60 (n=14), day 90 (n=13)) and RC9 (F; day 14 (n=29), day 28 (n=29), day 60 (n=28), day 90 (n=15)); Mixed effect analysis using Dunnett’s multiple comparison; Significance over day 14, when indicated. All data in Figure 3 are presented as mean ±S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001 .

[0074] Figure 4 shows enrichment of SC-islets by density gradient separation. (A) Schematic representation of the density gradient separation method. A linear density gradient was first prepared (not shown). S7 SC-islets cell clusters were then loaded on top of the gradient medium in a 50 ml conical tube that was subsequently subjected to centrifugation. Next, seven fractions of a similar volume were collected in new 50ml tubes, and further assessed for their composition. (B) Representation of the linear density (g / ml) gradient as calculated (theoretical) for each fraction, and experimentally validated by measurement using a density meter. (C) Representative images of a viability assay of cell clusters stained with FDA (live cells, green) / PI (dead cells, red) at 1 day (left) and 4 days (right) post density gradient separation. Scale bar, 200 pm. (D) Representative images of a dithizone (DTZ) staining of enriched SC-islet (left) and depleted SC-islet (right) fractions. (E) Proportion of DTZ+ tissue in each fraction, normalized to the amount of tissue (DNA content per fraction). Data were collected from 3 independent experiments (HUES8 (n=2), LUMC iPSC1 (n=1)). (F) Proportion of DTZ+ tissue in enriched SC-islets (pooled fractions 2, 3, 4, 5) and depleted SC- islets (fractions 1 , 6, 7), normalized to the amount of tissue (DNA content per fraction). Data were collected from 3 independent experiments (HUES8 (n=2), LUMC iPSC1 (n=1)). (G) Representative images of immunostaining for Synaptophysin (SYP; endocrine cells; green) and KRT19 (ductal cells; pink) in enriched SC-islets (left) and depleted SC-islets (middle); and a representative image of immunostaining of C-peptide (C-pep; green) and glucagon (GCG; pink) in enriched SC-islets (left); scale bar, 200 pm. (H) Electron microscopy image of a p cell from enriched SC-islets labeled with immunogold staining for insulin. Scale bars, 1 pm. (I) Proportion of DTZ+ tissue in each fraction, normalized to the amount of tissue (DNA content per fraction). Data were collected from 7 independent experiments (HUES8 (n=3), LUMC iPSC1 (n=4)). (J) Proportion of DTZ+ tissue in enriched S7 SC-islets (pooled fractions 2, 3, 4, 5) and depleted S7 SC-islets (fractions 1 , 6, 7), normalized to the amount of tissue (DNA content per fraction). Data were collected from 7 independent experiments (HUES8 (n=3), LUMC iPSC1 (n=4)). (K) UMAP plot indicating the cell populations in S7 SC-islets before (SC-islet) and after (Enriched SC-islet) density gradient separation (HUES8). (L) Frequency of the cell types present in S7 SC-islets before (SC-islet) and after (Enriched SC-islet) enrichment (HUES8). (M) Frequency of the endocrine and non-endocrine cells in S7 SC-islets before (SC-islet) and after (Enriched SC-islet) enrichment (HUES8).

[0075] Figure 5 shows that enriched SC-islets function well in vitro and in vivo. (A) Stimulation index (fold secretion of C-peptide concentration in high glucose over first low glucose) of S7 SC-islets (non-purified) and enriched SC-islets in seven independent experiments (HUES8 (n=5), RC9 (n= 1 ), LUMC iPSC1 (n=1)) after subsequent incubations with 1.67 mM, 20 mM and 1.67 mM glucose; Student paired two-tailed t test; Significance versus non-purified cells; (B) Mitochondrial properties indicating Oxygen Consumption Rate (OCR) normalized to protein content in enriched SC-islets (fraction 4), compared to S7 SC-islets; two independent experiments (HUES8 (n= 1 ), RC9 (n=1)). Multiple paired t-test; Significance to non-purified cells, if indicated. (C) Intracellular calcium ([Ca2+]) recordings upon incubations of enriched SC-islets or S7 SC-islets derived from HUES8 (n=1 , left) and RC9 (n=1 , right) with 1.67 mM, 20 mM glucose or 30 mM KCI. Relative fluorescence changes as a function of time, showing the average of cell response (top heatmap) and each line representing one cell (bottom heatmap). (D) Representative immunostainings for C-peptide (green) and Glucagon (GCG, pink) of enriched SC-islets at 100 days post-engraftment; Scale bar, 200 pM. An insert with a higher magnification is shown. Scale bar, 50 pM. (E) Maximum stimulated human C- peptide concentration in mice engrafted with (non-purified S7 SC-islets or enriched SC-islets derived from HUES8 (2 independent experiments, 6 mice per condition). (F) Stimulation index (fold change of C-peptide secretion in high glucose over average of basal secretion in the last 3 time points of first low glucose) of S7 SC-islets (non-purified) and enriched S7 SC-islets in 5 independent experiments (HUES8 (n=2), LUMC iPSC1 (n=3)) during dynamic perifusion with 1.67 mM to 20 mM glucose and 30 mM KCI; Student paired one-tailed t test; Significance versus average of 3 time points in first low glucose, when indicated. (G) Fold secretion of C-peptide concentration in KCI over first low glucose of S7 SC-islets (non-purified) and enriched S7 SC-islets in 5 independent experiments (HUES8 (n=2), LUMC iPSC1 (n=3)). Student paired one-tailed t test; Significance versus SC-islet (nonpurified), when indicated. (H) C-peptide content (ng) normalized to DNA content (pg) of S7 SC-islet non-purified) and enriched S7 SC-islets in 5 independent experiments (HUES8 (n=2), LUMC iPSC1 (n=3)). Student paired one-tailed t test; Significance versus SC-islet (non-purified), when indicated. (I) Maximum stimulated human C-peptide concentration in mice engrafted with S7 SC-islets (nonpurified) or enriched S7 SC-islets derived from HUES8 (2 independent experiments, 6 mice per condition up to 100 days and 3 mice per condition up to 170 days). Mixed effect analysis using Sidak's multiple comparisons test when comparing enriched SC-islets to SC-islets. Significance over SC-islets, when indicated. Mixed effect analysis using Dunnett's multiple comparisons test when comparing days post transplantation to day 14 within each group. Significance over day 14, when indicated. (J) Representative immunostainings for Synaptophysin (SYP; endocrine cells; green) and KRT19 (ductal cells; pink) (left), and C-peptide (green) and Glucagon (GCG, pink) (right) in SC-islets before (top) and after enrichment (bottom) at 170 days post-engraftment; Scale bar, 200 pM. An insert with a higher magnification is shown. Scale bar, 50 pM. All data in Figure 5 are presented as mean ±S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001 .

[0076] Figure 6. (A) Overview of the seven-stage differentiation protocol performed in a 3D culture system. (B) Stimulation index (fold change of C-peptide secretion in high glucose over average of basal secretion in the last 3 time points of first low glucose) during dynamic perfusion with 1 .67 mM to 20 mM glucose and 30 mM KCI in S7 SC-islets in 4 individual experiments (HUES8 (n=2), LUMC iPSC1 (n=2)). (C) Frequency of C-peptide (C-pep) and glucagon (GCG) positive cells in 5 cell lines (HUES8, RC9, LUMC iPSC1 , LUMC iPSC2, LUMC iPSC3), as assessed by flowcytometry. (D) Frequency of C-peptide (C-pep), glucagon (GCG), somatostatin (SST) and enterochromaffin (SLC18A1 ) positive cells by quantification of immunostaining images in 6 independent experiments (LUMC iPSC1 (n=6)). (E) A representative image of immunostaining for C-peptide (C-pep; green) and glucagon (GCG; pink), somatostatin (SST; white) in SC-islets (left), and for enterochromaffin (SLC18A1 ; green) cells (right); scale bar, 50 pm. (F) Stimulation index (fold change of C-peptide secretion in high glucose over average of basal secretion in the last 3 time points of first low glucose) during dynamic perfusion with 1.67 mM to 20 mM glucose and 30 mM KCI in S7 SC-islets in 11 individual experiments (HUES8 (n=3), LUMC iPSC1 (n=8)); Student paired one-tailed t test; Significance versus average of 3 time points in first low glucose, when indicated.

[0077] Figure 7. (A) Heatmap showing the average expression of selected genes associated with p cell identity (38) in p cells from donor islets (donor-p, n=3) and S7 SC-p cells derived from HUES8 (HUES8-P, n=2) or RC9 (RC9-P, n=3). (B) Heatmap showing the average expression of hallmark genes related to oxidative phosphorylation (MSigDB database) in p cells from donor islets (donor-p, n=3) and S7 SC-p cells derived from HUES8 (HUES8-P, n=2) or RC9 (RC9-P, n=3). (C) Heatmap showing the average expression of hallmark genes related to protein secretion (MSigDB database) in p cells from donor islets (donor-p, n=3) and S7 SC-p cells derived from HUES8 (HUES8-P, n=2) or RC9 (RC9-P, n=3).

[0078] Figure 8. (A and B) Stimulated human C-peptide concentration in mice engrafted with S7 SC-islets derived from HUES8 (A; day 14 (n=19), day 28 (n=19), day 60 (n=10), day 90 (n=9)) and RC9 (B; day 14 (n=33), day 28 (n=33), day 60 (n=29), day 90 (n= 14)), and at 0, 30 and 60 minutes post glucose injection. (C and D) Area under the curve (AUC) of the Figures 7A and B. Mixed effect analysis using Dunnett’s multiple comparison; Significance over day 14, when indicated. All data in Figure 8 are presented as mean ± S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001 .

[0079] Figure 9. (A) Purity as assessed by the percentage of dithizone (DTZ) positive cell clusters in each fraction; data were collected from 3 independent experiments (HUES8 (n=2), LUMC-iPSC1 (n=1)). (B) Tissue content as assessed by DNA content in each fraction, and normalized to the total amount of tissue collected in all fractions. Data are from 7 independent experiments (HUES8 (n=4), RC9 (n=2), LUMC-iPSC1 (n=1)). (C) Proportion of tissue collected in enriched SC-islets (pooled fractions 2-5) and depleted SC-islets (pooled fractions 1 , 6, 7). HUES8 (n=4), RC9 (n=2), LUMC iPSC1 (n=1). (D) Proportion of C-peptide content normalized to DNA (Figure 9B) from each fraction. HUES8 (n=4), RC9 (n=2), LUMC iPSC1 (n=1); Paired t-test; Significance to non-purified cells, if indicated. (E) Proportion of total C-peptide content in enriched SC-islets (pooled fractions 2-5) and depleted SC-islets (fractions 1 , 6 and 7). HUES8 (n=4), RC9 (n=2), LUMC iPSC1 (n=1)); Paired t- test; Significance to non-purified cells, if indicated. All data in Figure 9 are presented as mean ±S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001 .

[0080] Figure 10. (A and B) Stimulated human C-peptide concentration in mice engrafted with S7 SC-islets derived from HUES8 (A; day 14 (n=14), day 28 (n=14), day 60 (n=14), day 90 (n=13)) and RC9 (B; day 14 (n=29), day 28 (n=29), day 60 (n=28), day 90 (n=15)), at 0, 30 and 60 minutes post glucose injection. Student paired one-tailed t test; Significance versus time 0 (before glucose injection) when indicated. (C and D) Area under the curve (AUC) of Fig. 10A and B. Mixed effect analysis using Dunnett’s multiple comparisons; Significance over day 14, when indicated. All data in Figure 10 are presented as mean ± S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001.

[0081] Figure 11 . (A) Purity as assessed by the proportion of dithizone (DTZ) positive cell clusters in each fraction; Data were collected from 8 independent experiments (HUES8 (n=4), LUMC-iPSC1 (n=4)). (B) Tissue content as assessed by DNA content in each fraction, and normalized to the total amount of tissue collected in all fractions. Data are from 15 independent experiments (HUES8 (n=6), RC9 (n=2), LUMC-iPSC1 (n=7)). (C) C-peptide content normalized to DNA content in each fraction. Data are from 11 independent experiments (HUES8 (n=4), RC9 (n=2), LUMC iPSC1 (n=5)). (D) C- peptide content in enriched S7 SC-islets (Enriched, pooled fractions 2-5) and depleted S7 SC-islets (Depleted, fractions 1 , 6 and 7). Data are from 11 independent experiments (HUES8 (n=4), RC9 (n=2), LUMC iPSC1 (n=5)); Unpaired two-tailed t-test. (E) Purity as assessed by the frequency of dithizone (DTZ) positive cell clusters in enriched S7 SC-islets (pooled fractions 2-5) and depleted S7 SC-islets (pooled fractions 1 , 6, 7); Data were collected from 8 independent experiments (HUES8 (n=4), LUMC-iPSC1 (n=4)). Student paired one-tailed t test; Significance versus SC-islet (nonpurified). (F) C-peptide content in enriched S7 SC-islets (pooled fractions 2-5) and depleted S7 SC- islets (fractions 1 , 6 and 7) across individual cell lines (HUES8 (n=4), RC9 (n=2), LUMC iPSC1 (n=15)); Unpaired two-tailed t-test. (G) Tissue content in enriched S7 SC-islets (pooled fractions 2-5) and depleted S7 SC-islets (pooled fractions 1 , 6, 7). Data are from 15 independent experiments (HUES8 (n=6), RC9 (n=2), LUMC-iPSC1 (n=7)). Student unpaired two-tailed t test. (H) Quantification of immunostaining images for C-peptide (C-pep) and glucagon (GCG) in 3 independent experiments (HUES8 (n=1), LUMC iPSC1 (n=2)). Data are indicated as fold change of (C-pep+GCG) in enriched and depleted S7 SC-islets over S7 SC-islets (non-purified); One way ANOVA with Dunnett’s multiple comparison tests. Significance over SC-islets (non-purified) when indicated. (I) Quantification of immunostaining images for synaptophysin (SYP; endocrine cells) and KRT19 (ductal cells) in 3 independent experiments (HUES8 (n=1), LUMC iPSC1 (n=2)). Data are indicated as fold change of ratio (SYP / KRT19) in enriched and depleted S7 SC-islets over S7 SC- islets (non-purified); One way ANOVA with Dunnett’s multiple comparison tests. Significance over SC-islets (non-purified) when indicated. (J) Heatmap showing the average expression of selected genes associated with p cell identity and maturation in p cells from donor islets (donor-p, n=3) and SC-p cells derived from HUES8 (HUES8-P) in enriched S7 SC-islet and S7 SC-islet (non-purified). (K) Heatmap showing the average expression of genes related to p cell metabolic sensing and signaling including glucose sensing in p cells from donor islets (donor-p, n=3) and SC-p cells derived from HUES8 (HUES8-P, n=1) in enriched S7 SC-islet and S7 SC-islet (non-purified). (L) Heatmap showing the average expression of genes related to function and secretion, exocytosis, and non- canonical factors in p cells from donor islets (donor-p, n=3) and SC-p cells derived from HUES8 (HUES8-P, n=1) in enriched S7 SC-islet and S7 SC-islet (non-purified). (M to O) Violin plot representing the expression of selected genes (from panel K and L) related to glycolysis and TCA cycle (M), oxidative phosphorylation (N), and insulin secretion (O) in p cells from donor islets (donor- P, n=3) and SC-p cells derived from HUES8 (HUES8-P, n=1) in enriched S7 SC-islet and S7 SC-islet (non-purified). (P) Matrix plot indicated the marker genes used for cluster annotation. Expression scaled from 0 to 1 , based on maximum and minimum mean expression (HUES8, n=1). (Q) Frequency of the cell types present in S7 SC-islets before (SC-islet) and after (Enriched SC-islet) enrichment (HUES8, n=1). All data are presented as mean ±S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001.

[0082] Figure 12. (A and B) Stimulated human C-peptide concentration in mice engrafted with S7 SC-islets (non-purified) or enriched S7 SC-islets derived from HUES8 (2 independent experiments, 6 mice per condition up to 100 days and 3 mice per condition up to 170 days) at 0, 30 and 60 minutes post glucose injection. Student paired one-tailed t test; Significance versus time 0 (before glucose injection) when indicated. (C and D) Area under the curve (AUC) of Fig. 12A-B. Mixed effect analysis using Dunnett’s multiple comparison; Significance over day 14, when indicated. All data are presented as mean ± S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001 . (E) A representative image of immunostaining for synaptophysin (SYP; endocrine cells) and CK19 (ductal cells) positive cells (left, scale bar 200 pm; inserts, scale bar 50 pm) in the enriched S7 SC-islets engrafted in immunodeficient mice at day 0 post transplantation. A representative IHC staining for insulin in the enriched S7 SC-islets engrafted in immunodeficient mice at day 0 post transplantation (top right). (F) A representative image of immunostaining for C-peptide (C-pep) and MAFA positive cells in the enriched S7 SC-islets engrafted in immunodeficient mice at day 170 post transplantation.; scale bar 200 pm (left) and 50 pm (right). (G) Non-fasting blood glucose measurement (mM) over 170 days of engraftment of enriched S7 SC-islets and S7 SC-islets (non-purified). (H and I) Blood glucose measurement, following IPGTT (intraperitoneal glucose tolerance test) in mice engrafted with S7 SC-islets and enriched S7 SC-islets before glucose injection (Time 0) and at 15, 30, 60 and 120 minutes post glucose injection (2 independent experiments, 6 mice per condition up to 100 days and 3 mice per condition up to 168 days). (J and K) Area under the curve (AUC) of the Fig. 12H-I. Mixed effect analysis using Dunnett’s multiple comparison; Significance over day 14, when indicated. All data are presented as mean ± S.E.M. * P < 0.05, ** P < 0.01 , *** P < 0.001 . (L) A representative image of immunostaining for C-peptide (C-pep) and SLC18A1 positive cells in the enriched SC-islets engrafted in immunodeficient mice at day 170 post transplantation; scale bar 200 pm (left) and 50 pm (right).

[0083] Figure 13. C-peptide and tissue distribution following discontinuous density gradient separation. (A) Proportion of C-peptide content normalized to the DNA content (%) in enriched fractions (pooled fractions 3 and 4) and depleted fractions (pooled fractions 1 , 2, and 5). The data were collected from three individual experiments. (B) Proportion of the tissue collected in enriched fractions and depleted fractions. Xenetix is indicated as Xen. (C) C-peptide distribution across fractions. Values were normalized to DNA content. (D) Tissue distribution across fractions assessed by DNA content. All data in Figure 13 are presented as mean ± SEM. * = p < 0.05, ** = p < 0,01 , **** = P < 0,0001.

[0084] Further details of the figures are disclosed in the Examples below.

[0085] Detailed description of the invention

[0086] The present invention relates to a method of enriching cell clusters comprising ex vivo differentiated cells, the method comprising: a. subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and b. isolating the cell clusters from the first volume, thereby enriching the cell clusters.

[0087] The present invention also relates to a method of isolating cell clusters comprising ex vivo differentiated cells, the method comprising: a. subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and b. isolating the cell clusters from the first volume.

[0088] Hence, isolated or enriched cell clusters are obtained by the method of the invention.

[0089] The inventors have found a method of enriching or isolating cell clusters comprising ex vivo differentiated cells, where a suitable separation of cells is achieved resulting in one or more separate volumes comprising enriched or isolated cell clusters comprising target cells. Other separate volumes comprise for example cell clusters comprising less target cells, cell clusters comprising an undesired amount of non-target cells, or single cells. At the same time, the enriched or isolated cell clusters surprisingly remain substantially intact during the method, while also being intact and functional after performing the method. No noticeable effect on the biological activity or survival of the cells was observed. It appears that paracrine signaling and cell-cell interactions, and optional cell-matrix interactions, of the enriched or isolated cell clusters are substantially unaffected by the method of the invention. Interestingly, the inventors show that mice engrafted with enriched or isolated cell clusters according to the method of the invention have increased stimulated human C- peptide secretion compared to cell clusters that have not been enriched or isolated according to the method of the invention.

[0090] Suitably, the enriched or isolated cell clusters may comprise at least 95% viable cells.

[0091] Suitably, the enriched or isolated cell clusters may comprise at least 97% viable cells. Suitably, the enriched or isolated cell clusters may comprise at least 99% viable cells. Preferably, the viable cells may be determined by fluorescein diacetate (FDA) and propidium iodide (PI) staining, which is a commonly known method known in the art.

[0092] Suitably, the enriched or isolated cell clusters may be suitable for cell therapy or suitable for use in cell therapy.

[0093] Herein, the term “target cell” is understood to mean a cell having desired characteristics. The desired characteristics can be selected and determined in various way depending on the goals. For example, a specialized cell type may be desired, which exhibits functional characteristics closely relating to a fully differentiated cell. The thresholds of sufficient functionality or differentiation of the target cell may be set and determined accordingly by the skilled person. Functionality may for example be assessed via determination of produced compounds and / or by determination of certain expression markers. A sufficient functionality may for example be a level of secretion of therapeutic compounds, such as proteins, to a degree such that the cells or cell clusters are suitable for clinical application.

[0094] Herein, the term “non-target cell” is understood to mean a cell not having desired characteristics or not having the desired characteristics to a sufficient degree. Non-limiting examples of non-target cells include cells that are not (sufficiently) differentiated, do not secrete or express a low, insufficient level of therapeutic or functional compounds, or cells not present in clusters.

[0095] Herein, the term “volume” in the context of a density gradient medium comprising a volume, such as a first, second, third and / or further volume, according to the method (or kit) of the invention, is understood to mean that the volume is a portion of the density gradient medium with one or more specific densities or a gradient of densities. To clarify, if the volume has more than one specific discrete density, one may consider the volume as having multiple sub-volumes, each sub-volume having a specific density. After density gradient separation (e.g. after the centrifugation step) of for example cells or cell clusters, the volume contains cells or cell clusters with about the same density as the volume. If, for example, the volume has two sub-volumes of different densities, cells or cell clusters with a density between the density of the two sub-volumes may then appear in a so-called interphase between the two sub-volumes or they may appear near the borders of each sub-volume, for example as a band, phase, or cloud. Each different band, phase, or cloud may then also be known in the art as a fraction. The band, phase, or cloud may then be isolated from the first volume according to the invention and may be considered an isolated or enriched fraction. If the volume has a gradient of densities, cells or cell clusters may appear as different bands, phases, or clouds within the gradient after density gradient separation (e.g. after the centrifugation step). Multiple different fractions of cells or cell clusters may then be isolated from the first volume according to the invention and each may be considered an isolated or enriched fraction.

[0096] Density gradient separation

[0097] Density gradient separation is commonly used for the isolation of specific cell populations from whole blood. In density gradient separation, also called isopycnic, buoyant or equilibrium separation, cell clusters are separated on the basis of their density. Cell cluster size only influences the rate at which cell clusters move until their density is the same as the surrounding density gradient medium. Hence, the particular cell clusters isolated will depend on the density of the medium that is used.

[0098] Suitably, the first volume may have a density of from about 1 .0 g / ml to about 1 .3 g / ml, of from about 1.0 g / ml to about 1.2 g / ml, of from about 1.0 g / ml to about 1.1 g / ml, of from about 1.02 g / ml to about 1.20 g / ml, of from about 1.02 g / ml to about 1.14 g / ml, of from about 1.03 g / ml to about 1.30 g / ml, of from about 1.03 g / ml to about 1.15 g / ml, of from about 1.04 g / ml to about 1.20 g / ml, of from about 1.04 g / ml to about 1.16 g / ml, of from about 1.04 g / ml to about 1.12 g / ml, of from about 1.05 g / ml to about 1.20 g / ml, of from about 1.05 g / ml to about 1.15 g / ml, of from about 1.05 g / ml to about 1.13 g / ml, of from about 1.05 g / ml to about 1.11 g / ml, of from about 1.05 g / ml to about 1.09 g / ml, of from about 1 .053 g / ml to about 1 .088 g / ml, of from about 1 .053 g / ml to about 1 .097 g / ml, of from about 1.053 g / ml to about 1.081 g / ml, of from about 1.060 g / ml to about 1.075 g / ml, of from about 1 .060 g / ml to about 1 .080 g / ml, or of from about 1 .060 g / ml to about 1 .070 g / ml. Preferably, the first volume may have a density of from about 1 .053 g / ml to about 1 .088 g / ml. Preferably, the first volume may have a density of from about 1 .063 g / ml to about 1 .072 g / ml. Preferably, the first volume may have a density of from about 1 .065 g / ml to about 1 .075 g / ml. Preferably, the first volume may have a density of from about 1 .063 g / ml to about 1 .075 g / ml. Preferably, the first volume may have a density of from about 1 .0 g / ml to about 1 .3 g / ml.

[0099] Suitably, the first volume may have a single, discrete density, for example a density of 1 .09 g / ml. Suitably, the first volume may have multiple discrete densities, preferably contained in separate sub-volumes, for example a density of 1 .05 g / ml in a first sub-volume and a density of 1 .09 g / ml in a second sub-volume. This allows the separation of cell clusters having a density between 1 .05 g / ml and 1 .09 g / ml as these cell clusters may for example be located at an interphase between the first and second sub-volumes following density gradient separation.

[0100] Suitably, the first volume may comprise at least a first and a second sub-volume. Suitably, the first sub-volume may have a density of about 1 .0 g / ml, about 1 .01 g / ml, about 1 .02 g / ml, about 1 .03 g / ml, about 1 .04 g / ml, about 1 .05 g / ml, about 1 .06 g / ml, about 1 .07 g / ml, about 1 .08 g / ml, about 1 .09 g / ml, or about 1.1 g / ml. Suitably, the first sub-volume may have a density of about 1 .040 g / ml, about 1 .045 g / ml, about 1 .047 g / ml, about 1 .048 g / ml, about 1 .049 g / ml, about 1 .050 g / ml, about 1.051 g / ml, about 1.052 g / ml, about 1.053 g / ml, about 1.054 g / ml, about 1.055 g / ml, about 1.056 g / ml, about 1 .057 g / ml, about 1 .058 g / ml, about 1 .059 g / ml, about 1 .060 g / ml, about 1 .061 g / ml, about 1 .062 g / ml, about 1 .063 g / ml, about 1 .064 g / ml, about 1 .065 g / ml, about 1 .066 g / ml, about

[0101] 1 .067 g / ml, about 1 .068 g / ml, about 1 .069 g / ml, about 1 .070 g / ml, about 1 .071 g / ml, about 1 .072 g / ml, about 1 .073 g / ml, about 1 .074 g / ml, or about 1 .075 g / ml. Preferably, the first sub-volume may have a density of about 1 .05 g / ml. Preferably, the first sub-volume may have a density of about 1 .053 g / ml. Preferably, the first sub-volume may have a density of about 1 .063 g / ml. Preferably, the first sub-volume may have a density of about 1 .065 g / ml.

[0102] Suitably, the second sub-volume may have a density of about 1 .3 g / ml, about 1 .25 g / ml, about 1.2 g / ml, about 1.20 g / ml, about 1.19 g / ml, about 1.18 g / ml, about 1.17 g / ml, about 1.16 g / ml, about 1.15 g / ml, about 1.14 g / ml, about 1.13 g / ml , about 1.12 g / ml , about 1.11 g / ml, about 1.1 g / ml, about 1.10 g / ml, about 1.09 g / ml or about 1.08 g / ml. Suitably, the second sub-volume may have a density of about 1 .099 g / ml, about 1 .098 g / ml, about, 1 .097 g / ml, about 1 .096 g / ml, about 1 .095 g / ml, about 1 .094 g / ml, about 1 .093 g / ml, about 1 .092 g / ml, about 1 .091 g / ml, about 1 .090 g / ml, about 1 .089 g / ml, about 1 .088 g / ml, about 1 .087 g / ml, about 1 .086 g / ml, about 1 .085 g / ml, about

[0103] 1 .084 g / ml, about 1 .083 g / ml, about 1 .082 g / ml, about 1 .080 g / ml, about 1 .079 g / ml, about 1 .078 g / ml, about 1.077 g / ml, about 1.076 g / ml, or about 1.075 g / ml. Preferably, the second sub-volume may have a density of about 1.09 g / ml. Preferably, the second sub-volume may have a density of about 1 .088 g / ml. Preferably, the second sub-volume may have a density of about 1 .072 g / ml. Preferably, the second sub-volume may have a density of about 1 .075 g / ml.

[0104] Suitably, the first volume may comprise at least a first and a second sub-volume, wherein the first sub-volume has a density of about 1.053 g / ml and the second sub-volume has a density of about 1 .088 g / ml. Suitably, the first volume may comprise at least a first and a second sub-volume, wherein the first sub-volume has a density of about 1.063 g / ml and the second sub-volume has a density of about 1 .072 g / ml. Suitably, the first volume may comprise at least a first and a second sub-volume, wherein the first sub-volume has a density of about 1 .065 g / ml and the second subvolume has a density of about 1 .075 g / ml. Suitably, the first volume may comprise at least a first and a second sub-volume, wherein the first sub-volume has a density of about 1 .063 g / ml and the second sub-volume has a density of about 1 .075 g / ml.

[0105] A density gradient can be constructed by various methods known in the art. Several types of gradients can be employed. Examples of these types are: a discontinuous or segmented gradient, which can be formed by layering zones of density gradient medium of decreasing density on top of each other; a linear or continuous gradient, in which the density of the medium increases linearly with distance down the container used for separation; and an isokinetic gradient, which is constructed such that the increasing centrifugal force on the particle during centrifugation is always counterbalanced by the increasing viscous force and the decreasing “effective mass.”.

[0106] Herein, the full density gradient medium is used for the density gradient separation. The density gradient medium comprises at least the first and the second volume, but may optionally comprise a third and / or further volumes. Better separation and reduced loss of cell clusters may be achieved by employing a segmented density gradient. A segmented density gradient can be suitably designed to more selectively collect target cells in cell clusters having a specific density, while potentially also including one or more volumes with a density gradient that result in improved separation of non-target cells.

[0107] Suitably, the first volume may have a gradient of densities, for example a continuous density gradient, such as of from 1.1 g / ml to 1.3 g / ml. A continuous gradient for the first volume or the full density gradient medium may for example be created by layering multiple discrete (sub-)volumes having different densities on top of each other and allowing the (sub-)volumes to diffuse in a controlled manner. For example, the full density gradient medium may then be created to have a density gradient of from 0.9 g / ml to 2.0 g / ml, the density gradient medium further comprising a first volume having, for example, a density gradient of from 1.1 g / ml to 1.3 g / ml.

[0108] Suitably, the density gradient medium or the first volume may have a continuous, linear, discontinuous, segmented, or isokinetic density gradient, preferably a segmented density gradient. Suitably, the density gradient medium or the first volume may have a continuous or discontinuous density gradient.

[0109] Suitably, the first volume may have a gradient of densities that is within the range of from about 1 .0 g / ml to about 1 .3 g / ml, of from about 1 .0 g / ml to about 1 .2 g / ml, of from about 1 .0 g / ml to about 1.1 g / ml, of from about 1.02 g / ml to about 1.20 g / ml, of from about 1.02 g / ml to about 1.14 g / ml, of from about 1.03 g / ml to about 1.30 g / ml, of from about 1.03 g / ml to about 1.15 g / ml, of from about 1.04 g / ml to about 1.20 g / ml, of from about 1.04 g / ml to about 1.16 g / ml, of from about 1.04 g / ml to about 1.12 g / ml, of from about 1.05 g / ml to about 1.20 g / ml, of from about 1.05 g / ml to about 1.15 g / ml, of from about 1.05 g / ml to about 1.13 g / ml, of from about 1.05 g / ml to about 1.11 g / ml, of from about 1 .05 g / ml to about 1 .09 g / ml, of from about 1 .053 g / ml to about 1 .088 g / ml, of from about 1 .053 g / ml to about 1 .097 g / ml, of from about 1 .053 g / ml to about 1 .081 g / ml, of from about 1 .060 g / ml to about 1 .075 g / ml, of from about 1 .060 g / ml to about 1 .080 g / ml, or of from about 1 .060 g / ml to about 1 .070 g / ml. Preferably, the first volume may have a gradient of densities that is within the range of from about 1 .053 g / ml to about 1 .088 g / ml. Preferably, the first volume may have a gradient of densities that is within the range of from about 1.063 g / ml to about 1.072 g / ml. Preferably, the first volume may have a gradient of densities that is within the range of from about 1 .065 g / ml to about 1 .075 g / ml. Preferably, the first volume may have a gradient of densities that is within the range of from about 1 .063 g / ml to about 1 .075 g / ml. Preferably, the first volume may have a gradient of densities that is within the range of from about 1 .0 g / ml to about 1 .3 g / ml.

[0110] Isolating the cell clusters from the first volume may be facilitated by constructing a third volume. Hence, a better separation and enrichment can be obtained.

[0111] Suitably, the method may further comprise a third volume having a different density from the first and second volumes. Suitably, one of the second or third volumes may have a higher density than the first volume, and the other of the second or third volumes may have a lower density than the first volume.

[0112] The method of the invention can be suitably expanded by constructing additional volumes having specific other densities for enrichment of cell clusters comprising target cells. For example, this can be useful when the suspension comprises various cell clusters for enrichment that have different densities, while the suspension also contains cell clusters that are not to be enriched and have a density in between the cell clusters for enrichment.

[0113] The choice of density gradient medium depends on its ability to maintain the viability of the cell clusters. Typically, a medium that maintains the viability of the cell clusters outside the method of the invention also maintains the viability during the method. Various density gradient mediums are known in the art and can be selected accordingly by the skilled person.

[0114] Suitably, the density gradient medium or the first volume may comprise a cell gradient medium, a cell maintenance medium, a cell culture medium, a tissue preservation medium, and / or a colloid.

[0115] Examples of cell gradient media include, but are not limited to: Ficoll®, Ficoll-Paque®, Percoll®, Xenetix®, OptiPrep™, Histopaque®, media comprising monosaccharides such as sucrose, and media comprising polysaccharides. Examples of tissue preservation media include, but are not limited to: University of Wisconsin solution and Euro-Collins solution.

[0116] Suitably, the density gradient medium or the first volume may be about isosmotic, preferably about isosmotic to the cell clusters.

[0117] Suitably, the density gradient medium or the first volume may have on osmolarity of from about 260 mOsm to about 330 mOsm, of from about 260 mOsm to about 320 mOsm, of from about 260 mOsm to about 310 mOsm, of from about 270 mOsm to about 310 mOsm, of from about 270 mOsm to about 290 mOsm, of from about 290 mOsm to about 330 mOsm, of from about 290 mOsm to about 320 mOsm, of from about 290 mOsm to about 310 mOsm, of from about 300 mOsm to about 330 mOsm, or of from about 310 mOsm to about 330 mOsm. Preferably, the density gradient medium or the first volume may have on osmolarity of from about 260 mOsm to about 330 mOsm. Preferably, the density gradient medium or the first volume may have on osmolarity of from about 275 mOsm to about 295 mOsm.

[0118] Density gradient separation, i.e. subjecting a suspension comprising the cell clusters to density gradient separation according to the invention, is typically performed by centrifugation using centrifuge containers for cells, such as centrifuge tubes or buckets. Methods of centrifugation of cells are commonly known in the art and the skilled person can accordingly select suitable parameters (e.g. centrifugal force and time) and materials for density gradient separation that also assist in maintaining the viability of the cell clusters. In general, the present invention is for example not limited to any particular rotor type or centrifuge container. Centrifugation of cells is typically performed at low temperatures. Suitably, subjecting the suspension may be performed at from 0 °C to about 4 °C, 8 °C, or 12 °C. Preferably, subjecting the suspension may be performed at from 0 °C to about 8 °C.

[0119] Ex vivo differentiated cells

[0120] Current techniques provide the possibility of culturing undifferentiated or partially differentiated cells, e.g. stem cells, ex vivo. Such cells have a high proliferative capacity and can be differentiated ex vivo into various types of mature specialized target cells. The differentiated cells can be produced in clinically relevant quantities. Various methods are known in the art for differentiating specific types of stem cells into specific target types of differentiated cells and can suitably be used by a person skilled in the art.

[0121] Preferably, the ex vivo differentiated cells may comprise cells derived from stem cells. Suitably, the ex vivo differentiated cells may comprise cells derived from totipotent stem cells, pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), multipotent stem cells, oligopotent stem cells, embryonic stem cells (ESC), mesenchymal stem cells, epithelial stem cells, or organoids. Preferably, the ex vivo differentiated cells may comprise cells derived from iPSC.

[0122] Suitably, the ex vivo differentiated cells may comprise cells derived from mammalian cells, preferably human cells.

[0123] Suitably, the ex vivo differentiated cells may comprise endocrine cells. Suitably, the ex vivo differentiated cells may comprise thyroid follicular cells, thyroid parafollicular cells, parathyroid chief cells, parathyroid oxyphil cells, or enteroendocrine cells. Suitably, the ex vivo differentiated cells may comprise pancreatic enteroendocrine cells or pancreatic islet cells. Preferably, the ex vivo differentiated cells may comprise pancreatic islet cells. Suitably, the ex vivo differentiated cells may comprise alpha cells, beta cells, gamma cells (also known as pancreatic polypeptide cells), delta cells and / or epsilon cells. Preferably, the ex vivo differentiated cells may comprise alpha cells and beta cells. Preferably, the ex vivo differentiated cells may comprise beta cells. Preferably, the alpha cells produce, more preferably secrete, glucagon. Preferably, the beta cells produce, more preferably secrete, insulin. Preferably, the gamma cells produce, more preferably secrete, pancreatic polypeptide. Preferably, the delta cells produce, more preferably secrete, somatostatin. Preferably, the epsilon cells produce, more preferably secrete, ghrelin. Preferably, the thyroid follicular cells produce, more preferably secrete, thyroxine and / or triiodothyronine. Preferably, the thyroid parafollicular cells produce, more preferably secrete, calcitonin. Preferably, the parathyroid chief cells produce, more preferably secrete, parathyroid hormone. Preferably, the parathyroid oxyphil cells produce, more preferably secrete, parathyroid hormone-related protein and / or calcitriol.

[0124] Enriched cell clusters

[0125] A complete or full ex vivo differentiation of undifferentiated or partially differentiated cells, e.g. stem cells, is difficult to achieve. Suboptimal differentiation remains a problem, which results in heterogeneous cell populations comprising non-target cells with undesired characteristics, for example insufficiently differentiated cells or undifferentiated cells, which have insufficient functional or clinical efficacy, or may be tumorigenic. The method of the invention enriches for or isolates cell clusters comprising ex vivo differentiated cells, thereby preferably enriching for or isolating cell clusters comprising an as high as possible percentage of ex vivo differentiated cells. Moreover, certain undifferentiated cells may be easier to differentiate into certain differentiated cells than others.

[0126] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may substantially consist of ex vivo differentiated cells. Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may substantially consist of ex vivo differentiated cells derived from stem cells. Suitably, the cell clusters, preferably the enriched cell clusters, may substantially consist of ex vivo differentiated cells derived from totipotent stem cells, pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), multipotent stem cells, oligopotent stem cells, embryonic stem cells (ESC), mesenchymal stem cells, epithelial stem cells, or organoids. Preferably, the cell clusters, preferably the enriched or isolated cell clusters, may substantially consist of ex vivo differentiated cells derived from iPSC.

[0127] Ex vivo differentiated cells can be combined into cell clusters, which may occur spontaneously during differentiation or may be supported or enabled by various known cell culture techniques or materials. The combination of differentiated cells into clusters provides for cell-cell interactions, optional cell-matrix interactions, and increased paracrine signalling, all of which support key functional, structural, and biological features of the differentiated cells in the context of their role in their intended in vivo tissue or organ. The cell clusters may therefore mimic an organ and show some degree of organotypic functionality.

[0128] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may be organotypic cell clusters. Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may be organotypic endocrine tissue. Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may be organotypic pancreatic islets, organotypic thyroid follicles, or organotypic parathyroid follicles. Preferably, the enriched or isolated cell clusters may be organotypic pancreatic islets.

[0129] Ex vivo differentiated cells can be combined into cell clusters by various ways known in the art. Cell culture of cell clusters for subsequent application in the method of the invention is preferably performed by three-dimensional (3D) cell culture, optionally wherein prior to 3D cell culture, the cells, or their progeny, comprised in the cell clusters have been cultured by two-dimensional (2D) cell culture. 3D cell culture permits cells to interact and grow in all three dimensions as opposed to classical 2D, or monolayer, cell culture. 3D cell culture more closely mimics in vivo growth conditions and enables improved paracrine signaling, and cell-cell and optional cell-matrix interactions. Advantageously, freely floating cell aggregates or suspensions of cell clusters may be formed via cell 3D culture. Methods and materials for 3D cell culture are well-known in the art. 3D cell culture can be performed by using supporting materials (also known as scaffolds) or without using supporting materials (also known as scaffold-free culture). Spheroids and organoids are typical examples of 3D cultures of cell clusters. Examples of scaffold-based 3D culture include, but are not limited to: using hydrogels, such as animal (cell) extracellular matrix extract hydrogels (e.g. Matrigel®), protein hydrogels, (poly)peptide hydrogels, or polymer (e.g. polysaccharide) hydrogels; or structural scaffolds comprising a suitable substrate material, such as in the form of microbeads, 3D printed substrates, or fibers. Certain scaffolds of scaffold-based 3D cultures may be removed while substantially maintaining the 3D structure of the cell clusters grown on or in the scaffold. As such, a suspension of cell clusters may be obtained. Examples of scaffold-free 3D culture include, but are not limited to: using low adhesion plates, hanging drop plates, micropatterned surfaces, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting, to create freely floating cell aggregates or suspensions of cell clusters, typically referred to as spheroids.

[0130] Suitably, the method according to the invention may further comprise obtaining the suspension, preferably comprising 3D culturing the cell clusters. Suitably, the method according to the invention may further comprise obtaining the suspension, preferably wherein the obtaining comprises 3D culturing the cell clusters. To clarify, here the cell clusters have undergone 3D cell culture prior to being obtained for use in the method according to the invention. Suitably, the method according to the invention may further comprise obtaining the suspension, wherein the obtaining comprises 3D culturing the cell clusters prior to being obtained. Suitably, 3D culturing the cell clusters may comprise suspension culturing, perfusion culturing and / or dynamic culturing. Suitably, 3D culturing the cell clusters may result in the formation of cell clusters.

[0131] Suitably, obtaining the suspension may comprise isolating the cell clusters from a 2D or 3D cell culture. Suitably, isolating the cell clusters may comprise not performing cell dissociation. Suitably, not performing cell dissociation may comprise not using cell dissociation buffer or cell dissociation enzymes. Suitably, isolating the cell clusters may comprise not using enzymes. Suitably, isolating the cell clusters may be enzyme-free. Suitably, obtaining the suspension may comprise not using enzymes. Suitably, obtaining the suspension may be enzyme-free. Preferably, the enzymes are enzymes that disrupt a physical connection between cells, between cells and extracellular matrix, and / or between cells and solid cell culture substrate. Such enzymes are typically known in the art as cell dissociation enzymes.

[0132] Suitably, the method according to the invention may be antibody-free and / or enzyme-free. Suitably, isolating the cell clusters may be antibody-free and / or enzyme-free.

[0133] As set out above, the combination of differentiated cells into clusters provides advantageous effects for and of the differentiated cells. Disrupting cell-cell interactions and optionally cell-matrix interactions therefore reduces these advantageous effects. Disrupting cell-cell interactions and cellmatrix interactions typically occurs when cell cultures are cultured, propagated, or processed (e.g. for clinical use), in particular when cells are detached from a scaffold or substrate. In contrast, the present invention may suitably and advantageously forego use of any cell dissociation methods or materials. For example, by 3D culturing the cell clusters comprising ex vivo differentiated cells (immediately) prior to density gradient separation according to the invention, preferably via a 3D scaffold-free culture, or when a scaffold is used for the 3D culture by substantially maintaining the 3D structure of the cell clusters grown on or in the scaffold.

[0134] Thus, the present invention also provides a method of enriching cell clusters comprising ex vivo differentiated cells, the method comprising: a. obtaining a suspension comprising the cell clusters, wherein the obtaining comprises 3D culturing the cell clusters prior to being obtained, optionally wherein the obtaining comprises isolating the cell clusters from a 3D cell culture and not performing cell dissociation; b. subjecting the suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and c. isolating the cell clusters from the first volume, thereby enriching the cell clusters.

[0135] In addition, the present invention also provides method of isolating cell clusters comprising ex vivo differentiated cells, the method comprising: a. obtaining a suspension comprising the cell clusters, wherein the obtaining comprises 3D culturing the cell clusters prior to being obtained, optionally wherein the obtaining comprises isolating the cell clusters from a 3D cell culture and not performing cell dissociation; b. subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and c. isolating the cell clusters from the first volume.

[0136] Also, the invention provides a method of enriching cell clusters comprising ex vivo differentiated cells, the method comprising: a. culturing the cell clusters in a three-dimensional (3D) cell culture; b. obtaining a suspension comprising the cell clusters; c. subjecting the suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and d. isolating the cell clusters from the first volume, thereby enriching the cell clusters.

[0137] Also, the invention provides a method of isolating cell clusters comprising ex vivo differentiated cells, the method comprising: a. culturing the cell clusters in a three-dimensional (3D) cell culture; b. obtaining a suspension comprising the cell clusters; c. subjecting the suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and d. isolating the cell clusters from the first volume. The functionality of the cell clusters can be enhanced by other cells. For example, the cell clusters may comprise a vascular structure. The cell clusters may comprise several types of differentiated cells, all, some, or one of which may be derived from stem cells. During or before ex vivo culture of the cell clusters other cells may be added.

[0138] Suitably, the ex vivo differentiated cells may comprise organoids, mesenchymal cells, endothelial cells, or epithelial cells.

[0139] Suitably, the enriched or isolated cell clusters may be substantially spherical or substantially spheroidal.

[0140] During density gradient separation, cell cluster size only influences the rate at which cell clusters move until their density is the same as the surrounding density gradient medium.

[0141] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may have a mean diameter of at least about 20 pm, of at least about 50 pm, of at least about 100 pm, of at least about 200 pm, of at least about 300 pm, of at least about 500 pm, of at least about 700 pm, or of at least about 1 mm. Preferably, the cell clusters, preferably the enriched or isolated cell clusters, may have a mean diameter of at least about 20 pm. Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may have a mean diameter of from about 20 pm to about 6 mm, of from about 20 pm to about 200 pm, of from about 20 pm to about 400 pm, of from about 20 pm to about 800 pm, of from about 40 pm to about 200 pm, of from about 40 pm to about 300 pm, of from about 40 pm to about 600 pm, of from about 40 pm to about 900 pm, of from about 100 pm to about 200 pm, of from about 100 pm to about 300 pm, of from about 100 pm to about 400 pm, of from about 100 pm to about 600 pm, of from about 100 pm to about 1 .2 mm, of from about 100 pm to about 2 mm, of from about 200 pm to about 400 pm, of from about 200 pm to about 600 pm, of from about 200 pm to about 800 pm, of from about 200 pm to about 1 mm, of from about 200 pm to about 2 mm, of from about 200 pm to about 3 mm, of from about 400 pm to about 800 pm, of from about 400 pm to about 2 mm, of from about 400 pm to about 4 mm, of from about 600 pm to about 1 mm, of from about 600 pm to about 1 .5 mm, of from about 600 pm to about 2.5 mm, of from about 600 pm to about 3 mm, of from about 1 mm to about 2 mm, of from about 1 mm to about 3 mm, of from about 1 mm to about 4 mm, of from about 1 mm to about 5 mm, of from about 2 mm to about 3 mm, of from about 2 mm to about 4 mm, of from about 2 mm to about 5 mm, of from about 2 mm to about 6 mm, of from about 3 mm to about 5 mm, or of from about 3 mm to about 7 mm. Preferably, the cell clusters, preferably the enriched or isolated cell clusters, may have a mean diameter of from about 20 pm to about 6 mm.

[0142] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may each have at least about 20 cells, at least about 50 cells, at least about 100 cells, at least about 200 cells, at least about 500 cells, at least about 1000 cells, at least about 2000 cells, or at least about 3000 cells. Preferably, the cell clusters, preferably the enriched or isolated cell clusters may each have at least about 20 cells. Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may each have of from about 20 to about 10000 cells, of from about 20 to about 7000 cells, of from about 20 to about 5000 cells, of from about 20 to about 2000 cells, of from about 20 to about 1000 cells, of from about 20 to about 500 cells, of from about 20 to about 200 cells, of from about 20 to about 100 cells, of from about 100 to about 10000 cells, of from about 100 to about 10000 cells, of from about 100 to about 8000 cells, of from about 100 to about 6000 cells, of from about 100 to about 4000 cells, of from about 100 to about 2000 cells, of from about 100 to about 1000 cells, of from about 100 to about 500 cells, of from about 200 to about 10000 cells, of from about 200 to about 8000 cells, of from about 200 to about 4000 cells, of from about 200 to about 2000 cells, of from about 200 to about 1000 cells, of from about 400 to about 10000 cells, of from about 400 to about 6000 cells, of from about 400 to about 4000 cells, of from about 400 to about 2000 cells, of from about 1000 to about 10000 cells, of from about 1000 to about 7000 cells, or of from about 1000 to about 4000 cells. Preferably, the cell clusters, preferably the enriched or isolated cell clusters, may each have of from about 20 to about 10000 cells

[0143] Suitably, the enriched cell clusters move until their density is the same as the surrounding density gradient medium, wherein the density gradient medium may have a density of from about 1.0 g / ml to about 1.3 g / ml, of from about 1.0 g / ml to about 1.2 g / ml, of from about 1.0 g / ml to about 1.1 g / ml, of from about 1.02 g / ml to about 1.20 g / ml, of from about 1.02 g / ml to about 1.14 g / ml, of from about 1.03 g / ml to about 1.30 g / ml, of from about 1.03 g / ml to about 1.15 g / ml, of from about 1.04 g / ml to about 1.20 g / ml, of from about 1.04 g / ml to about 1.16 g / ml, of from about 1.04 g / ml to about 1.12 g / ml, of from about 1.05 g / ml to about 1.20 g / ml, of from about 1.05 g / ml to about 1.15 g / ml, of from about 1.05 g / ml to about 1.13 g / ml, of from about 1.05 g / ml to about 1.11 g / ml, of from about 1 .05 g / ml to about 1 .09 g / ml, of from about 1 .053 g / ml to about 1 .088 g / ml, of from about 1 .053 g / ml to about 1 .097 g / ml, of from about 1 .053 g / ml to about 1 .081 g / ml, of from about 1 .060 g / ml to about 1 .075 g / ml, of from about 1 .060 g / ml to about 1 .080 g / ml, or of from about 1 .060 g / ml to about 1 .070 g / ml. Preferably, the density gradient medium may have a density of from about 1 .0 g / ml to about 1 .3 g / ml. Preferably, the density gradient medium may have a density of from about 1 .053 g / ml to about 1 .088 g / ml. Preferably, the density gradient medium have a density of from about 1 .063 g / ml to about 1.072 g / ml. Preferably, the density gradient medium may have a density of from about 1.065 g / ml to about 1.075 g / ml. Preferably, the density gradient medium may have a density of from about 1 .063 g / ml to about 1 .075 g / ml.

[0144] The method of the invention enriches for or isolates cell clusters comprising ex vivo differentiated cells, thereby preferably enriching for or isolating cell clusters comprising an as high as possible percentage of ex vivo differentiated cells.

[0145] Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may comprise endocrine cells. Suitably the cell clusters, preferably the enriched or isolated cell clusters, may comprise thyroid follicular cells, thyroid parafollicular cells, parathyroid chief cells, parathyroid oxyphil cells, or enteroendocrine cells. Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may comprise pancreatic enteroendocrine cells or pancreatic islet cells. Preferably, the cell clusters, preferably the enriched or isolated cell clusters, may comprise pancreatic islet cells. Suitably, the cell clusters, preferably the enriched or isolated cell clusters, may comprise alpha cells, beta cells, gamma cells (also known as pancreatic polypeptide cells), delta cells and / or epsilon cells. Preferably, the cell clusters, preferably the enriched or isolated cell clusters, may comprise alpha cells and beta cells. Preferably, cell clusters, preferably the enriched or isolated cell clusters, may comprise beta cells. Preferably, the alpha cells produce, more preferably secrete, glucagon. Preferably, the beta cells produce, more preferably secrete, insulin. Preferably, the gamma cells produce, more preferably secrete, pancreatic polypeptide. Preferably, the delta cells produce, more preferably secrete, somatostatin. Preferably, the epsilon cells produce, more preferably secrete, ghrelin. Preferably, the thyroid follicular cells produce, more preferably secrete, thyroxine and / or triiodothyronine. Preferably, the thyroid parafollicular cells produce, more preferably secrete, calcitonin. Preferably, the parathyroid chief cells produce, more preferably secrete, parathyroid hormone. Preferably, the parathyroid oxyphil cells produce, more preferably secrete, parathyroid hormone-related protein and / or calcitriol.

[0146] Suitably, the enriched or isolated cell clusters may not comprise exocrine cells.

[0147] Suitably, the enriched or isolated cell clusters may each comprise at least 55% endocrine cells. Suitably, the enriched or isolated cell clusters may each comprise at least 60%, at least 65%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, or at least 80% endocrine cells. Suitably, the enriched or isolated cell clusters may each comprise at most 15% non-endocrine cells. Suitably, the enriched or isolated cell clusters may each comprise at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1% non-endocrine cells.

[0148] Suitably, the enriched or isolated cell clusters may each comprise at least 40% C-peptide or insulin secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% C-peptide secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% insulin secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise at least of from 40% to 95% C-peptide or insulin secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise at least of from 40% to 95%, of from 40% to 90%, of from 40% to 85%, of from 40% to 80%, of from 50% to 95%, of from 50% to 90%, of from 50% to 85%, of from 50% to 80%, of from 55% to 95%, of from 55% to 90%, of from 55% to 85%, of from 55% to 80%, of from 60% to 95%, of from 60% to 90%, of from 60% to 85%, of from 60% to 80%, of from 65% to 95%, of from 65% to 90%, of from 65% to 85%, of from 65% to 80%, of from 70% to 95%, of from 70% to 85%, or of from 70% to 80% C- peptide secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise at least of from 40% to 95%, of from 40% to 90%, of from 40% to 85%, of from 40% to 80%, of from 50% to 95%, of from 50% to 90%, of from 50% to 85%, of from 50% to 80%, of from 55% to 95%, of from 55% to 90%, of from 55% to 85%, of from 55% to

[0149] 80%, of from 60% to 95%, of from 60% to 90%, of from 60% to 85%, of from 60% to 80%, of from

[0150] 65% to 95%, of from 65% to 90%, of from 65% to 85%, of from 65% to 80%, of from 70% to 95%, of from 70% to 85%, or of from 70% to 80% insulin secreting cells of the total cell number of the respective cell cluster.

[0151] Suitably, the enriched or isolated cell clusters may comprise at least 40% C-peptide or insulin secreting cells. Suitably, the enriched or isolated cell clusters may comprise at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% C-peptide secreting cells. Suitably, the enriched or isolated cell clusters may comprise at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% insulin secreting cells. Suitably, the enriched or isolated cell clusters may comprise at least of from 40% to 95% C-peptide or insulin secreting cells. Suitably, the enriched or isolated cell clusters may comprise at least of from 40% to 95%, of from 40% to 90%, of from 40% to 85%, of from 40% to 80%, of from 50% to 95%, of from 50% to 90%, of from 50% to 85%, of from 50% to 80%, of from 55% to 95%, of from 55% to 90%, of from 55% to

[0152] 85%, of from 55% to 80%, of from 60% to 95%, of from 60% to 90%, of from 60% to 85%, of from

[0153] 60% to 80%, of from 65% to 95%, of from 65% to 90%, of from 65% to 85%, of from 65% to 80%, of from 70% to 95%, of from 70% to 85%, or of from 70% to 80% C-peptide secreting cells. Suitably, the enriched or isolated cell clusters may comprise at least of from 40% to 95%, of from 40% to 90%, of from 40% to 85%, of from 40% to 80%, of from 50% to 95%, of from 50% to 90%, of from 50% to 85%, of from 50% to 80%, of from 55% to 95%, of from 55% to 90%, of from 55% to 85%, of from 55% to 80%, of from 60% to 95%, of from 60% to 90%, of from 60% to 85%, of from 60% to 80%, of from 65% to 95%, of from 65% to 90%, of from 65% to 85%, of from 65% to 80%, of from 70% to 95%, of from 70% to 85%, or of from 70% to 80% insulin secreting cells.

[0154] Suitably, the enriched or isolated cell clusters may each comprise at least 2%, at least 5%, at least 7%, at least 9%, at least 10%, at least 12%, at least 15%, or at least 17% glucagon secreting cells of the total cell number of the respective cell cluster. Preferably, the enriched or isolated cell clusters may each comprise at least 2% glucagon secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise of from 5% to 25%, of from 5% to 20%, of from 5% to 15%, of from 5% to 10%, of from 10% to 25%, of from 10% to 20%, or of from 15% to 25% glucagon secreting cells of the total cell number of the respective cell cluster.

[0155] Suitably, the enriched or isolated cell clusters may comprise at least 2%, at least 5%, at least 7%, at least 9%, at least 10%, at least 12%, at least 15%, or at least 17% glucagon secreting cells. Preferably, the enriched or isolated cell clusters may comprise at least 2% glucagon secreting cells. Suitably, the enriched or isolated cell clusters may comprise of from 5% to 25%, of from 5% to 20%, of from 5% to 15%, of from 5% to 10%, of from 10% to 25%, of from 10% to 20%, or of from 15% to 25% glucagon secreting cells.

[0156] Suitably, the enriched or isolated cell clusters may each comprise at least 70%, at least 72%, at least 74%, at least 75%, at least 77%, at least 79%, at least 80%, at least 82%, at least 85%, or at least 87%, thyroid hormone secreting cells of the total cell number of the respective cell cluster. Preferably, the enriched or isolated cell clusters may each comprise at least 70% thyroid hormone secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise of from 70% to 99%, of from 70% to 95%, of from 70% to 90%, of from 70% to 85%, of from 70% to 80%, of from 75% to 99%, of from 75% to 95%, of from 75% to 90%, of from 75% to 85%, of from 80% to 99%, of from 80% to 95%, or of from 80% to 90%, thyroid hormone secreting cells of the total cell number of the respective cell cluster. Preferably, the thyroid hormone secreting cells comprise triiodothyronine (T3) and / or thyroxine (T4) secreting cells.

[0157] Suitably, the enriched or isolated cell clusters may comprise at least 70%, at least 72%, at least 74%, at least 75%, at least 77%, at least 79%, at least 80%, at least 82%, at least 85%, or at least 87%, thyroid hormone secreting cells. Preferably, the enriched or isolated cell clusters may comprise at least 70% thyroid hormone secreting cells. Suitably, the enriched or isolated cell clusters may comprise of from 70% to 99%, of from 70% to 95%, of from 70% to 90%, of from 70% to 85%, of from 70% to 80%, of from 75% to 99%, of from 75% to 95%, of from 75% to 90%, of from 75% to 85%, of from 80% to 99%, of from 80% to 95%, or of from 80% to 90%, thyroid hormone secreting cells. Preferably, the thyroid hormone secreting cells comprise triiodothyronine (T3) and / or thyroxine (T4) secreting cells.

[0158] Suitably, the enriched or isolated cell clusters may each comprise at least 5%, at least 7%, at least 10%, at least 12%, or at least 15% calcitonin secreting cells of the total cell number of the respective cell cluster. Suitably, the enriched or isolated cell clusters may each comprise of from 5% to 25%, of from 5% to 20%, of from 5% to 15%, of from 7% to 25%, of from 7% to 20%, of from 10% to 25%, or of from 10% to 20%, calcitonin secreting cells of the total cell number of the respective cell cluster.

[0159] Suitably, the enriched or isolated cell clusters may comprise at least 5%, at least 7%, at least 10%, at least 12%, or at least 15% calcitonin secreting cells. Suitably, the enriched or isolated cell clusters may comprise of from 5% to 25%, of from 5% to 20%, of from 5% to 15%, of from 7% to 25%, of from 7% to 20%, of from 10% to 25%, or of from 10% to 20%, calcitonin secreting cells.

[0160] Herein, the term “purity” is understood to mean the percentage of desired or target cells out of the total number of isolated or enriched cells. Suitably, the desired or target cells may consist of live cells. Suitably, the total number of isolated or enriched cells may consist of live cells. Suitably, the total number of isolated cells may comprise live, dead, and dying cells.

[0161] Suitably, the enriched or isolated cell clusters may have a purity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Preferably, the enriched or isolated cell clusters may have a purity of at least 70%.

[0162] Suitably, the cell clusters may comprise target cells. Suitably, the cell clusters may comprise non-target cells. Suitably, the isolated or enriched cell clusters may comprise target cells. Suitably, the enriched cell clusters may comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% target cells of the total number of enriched cells.

[0163] Suitably, the isolated cell clusters may comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least

[0164] 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0165] 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least

[0166] 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% target cells of the total number of isolated cells.

[0167] Purity can be assessed by various means known in the art and depends on the desired characteristics of the target cells or cell clusters. The skilled person can select suitable methods for determining purity accordingly. For example, purity may be assessed via FACS or MACS, preferably performed on a sample of the enriched or isolated cell clusters. The threshold for the sufficient interaction or FACS or MACS signal can be set accordingly by the skilled person. Purity may also be assessed by adding compounds to the cells and determining if the interaction of the enriched cell clusters with the compounds is sufficient. Non-limiting examples include functional transport assays, fluorescent assays, binding assays, and staining assays, for example using compounds that stain or bind to endocrine cells or specific endocrine cells, such as dithizone for insulin producing cells. Dithizone (DTZ) is a zinc-chelating agent known to selectively stain pancreatic beta cells because of their high zinc content.

[0168] Suitably, the enriched cell clusters may comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least

[0169] 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0170] 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least

[0171] 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% dithizone positive cells of the total number of enriched cells. Preferably, the enriched cell clusters may comprise at least 50% dithizone positive cells of the total number of enriched cells. Preferably, the enriched cell clusters may comprise at least 70% dithizone positive cells of the total number of enriched cells.

[0172] Suitably, the isolated cell clusters may comprise at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

[0173] 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least

[0174] 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% dithizone positive cells of the total number of isolated cells. Preferably, the isolated cell clusters may comprise at least 50% dithizone positive cells of the total number of isolated cells. Preferably, the isolated cell clusters may comprise at least 70% dithizone positive cells of the total number of isolated cells.

[0175] A key property of mature pancreatic beta cells is their capacity to respond to a glucose challenge by secreting C-peptide. Elevating C-peptide secretion to a high glucose challenge and returning to the basal secretion in the subsequent exposure to low glucose are key features of beta cell function. Glucose stimulation index is defined as [ratio of C-peptide secreted in a solution comprising high glucose (e.g. 20 mM) to a solution comprising low glucose (e.g. 1.67 mM)]. A detailed preferred method for determining glucose-stimulated C-peptide secretion is disclosed in the Examples below. Suitably, glucose stimulation index of cell clusters, preferably enriched or isolated cell clusters, may be determined by the following method, comprising the following steps, wherein steps a to g are performed in sequential order: a. obtaining 40 cell clusters, preferably enriched or isolated cell clusters, more preferably enriched or isolated cell clusters according to the invention; b. equilibrating the enriched or isolated cell clusters in 1 .5 ml Krebs-Ringer Bicarbonate HEPES buffer (KRBH) comprising 1 .67 mM glucose for 90 min at 37 °C; c. subjecting the enriched or isolated cell clusters to 1 .5 ml KRBH comprising 1 .67 mM glucose for 60 min at 37 °C and collecting the supernatant; d. subjecting the enriched or isolated cell clusters to 1 .5 ml KRBH comprising 20 mM glucose for 60 min at 37 °C and collecting the supernatant; e. subjecting the enriched or isolated cell clusters to 1 .5 ml KRBH comprising 1 .67 mM glucose for 60 min at 37 °C and collecting the supernatant; f. subjecting the enriched or isolated cell clusters to 1 .5 ml KRBH comprising 30 mM KCI for 15 min at 37 °C and collecting the supernatant; g. collecting the enriched or isolated cell clusters and lysing the enriched or isolated cell clusters; h. determining the amount of C-peptide in the supernatants and the lysed enriched or isolated cell clusters using ELISA, preferably Human Ultrasensitive C-peptide ELISA (Mercodia, catalog no. 10-1141-01); i. determining the amount of DNA in the lysed enriched or isolated cell clusters, preferably using Picogreen assay (Thermo Fisher, P7589); j. normalizing the amount of C-peptide in the supernatants to total C-peptide content and normalizing the total amount of C-peptide to the amount of DNA; k. calculating the glucose stimulation index as a ratio of C-peptide in the supernatant in step d relative to the C-peptide in the supernatant in step c. Herein, Krebs-Ringer Bicarbonate HEPES buffer (KRBH) is a solution in water comprising 115mM NaCI, 5mM KCI, 24mM NaHCOs, 2.2mM CaCl2, 1 mM MgCl2, 0.2% human serum albumin, and 20mM HEPES, pH 7.4.

[0176] Suitably, the enriched or isolated cell clusters may have a glucose stimulation index of at least 1 .0, at least 1 .1 , at least 1 .2, at least 1 .3, at least 1 .4, at least 1 .5, at least 1 .6, at least 1 .7, at least 1 .8, at least 1 .9, at least 2.0, at least 2.1 , at least 2.2, at least 2.3, at least 2.4, or at least 2.5. Preferably, the enriched or isolated cell clusters may have a glucose stimulation index of at least 1 .2.

[0177] Pharmaceutical compositions

[0178] In one aspect the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the enriched or isolated cell clusters obtained by the method according to the invention.

[0179] An isolated or enriched cell cluster described herein may be provided as part of a pharmaceutical composition. Advantageously, such compositions may be administered to a human subject in need thereof (as described elsewhere herein).

[0180] Suitably, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0181] Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune suppressing agents, and optionally other therapeutic agents or compounds.

[0182] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e. , the material may be administered to an individual along with the isolated or enriched cell cluster without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0183] Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. enriched cell cluster as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, and without limitation, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, and ethanol.

[0184] Adjuvants are pharmacological and / or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art. Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0185] Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

[0186] In one example, the pharmaceutical composition comprises an isolated or enriched cell cluster, wherein the pharmaceutical composition is formulated as a cell therapy product (i.e. a composition that it used to place cells into the body of an individual to replace or repair diseased or damaged cells or tissue, or to modulate the function of the individual’s cells via expression of compounds (e.g. proteins) by the placed cells or via direct interaction of the placed cells).

[0187] T reatment of a subject

[0188] The cell therapies described herein can be used in treatment strategies in which cells are injected or otherwise transplanted into a patient to effectuate a medicinal effect.

[0189] In some examples, the cell clusters that are administered to the subject are allogeneic. In other examples, the cell clusters that are administered to the subject are autologous.

[0190] In an aspect the invention provides enriched or isolated cell clusters obtained by the method according to the invention, or the pharmaceutical composition according to the invention, for use as a medicament.

[0191] In another aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency, comprising obtaining enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, and administering to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition. Suitably, the subject may be a mammal. Suitably, the mammal may be a human.

[0192] In another aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency, comprising administering enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition.

[0193] A therapeutically effective amount herein refers to an amount sufficient to reduce the severity and / or duration of a defect, disorder, disease, deficiency, or a symptom thereof. Progression, development, or onset of the defect, disorder, disease, deficiency may thereby be reduced or prevented. The amount of enriched or isolated cell clusters or the pharmaceutical composition according to the invention actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound(s) administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0194] As used herein, the terms “treat”, “treating” and "treatment" are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom.

[0195] As used herein the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e. a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention.

[0196] The compositions described herein can be administered to the subject by any conventional route, including injection, implantation, or by gradual infusion over time. The administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.

[0197] The compositions described herein may be in any form suitable for the above modes of administration. For example, compositions comprising cell clusters may in any form be suitable for infusion or implantation. As further examples, suitable forms for parenteral injection (including, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion. Alternatively, the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery. The identification of suitable dosages of the compositions of the invention is well within the routine capabilities of a person of skill in the art.

[0198] The compositions described herein are for administration in an effective amount. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient / subject. For example, the suitable dosage of the composition of the invention for a given patient / subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the composition of the invention for example severity and type of haematological malignancy, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom and the overall condition of the patient / subject. Effective dosages may be determined by either in vitro or in vivo methods.

[0199] The pharmaceutical compositions described herein are advantageously presented in unit dosage form. In another aspect the invention provides enriched or isolated cell clusters obtained by the method according to the invention or the pharmaceutical composition according to the invention, for use in the treatment of a defect, disorder, disease, or deficiency of endocrine cells.

[0200] In one aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency of endocrine cells, comprising obtaining enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, and administering to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition.

[0201] In one aspect the invention provides a method of treating a subject that suffers from a defect, disorder, disease, or deficiency of endocrine cells, comprising administering enriched or isolated cell clusters according to the method according to the invention, or the pharmaceutical composition according to the invention, to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition.

[0202] Suitably, the defect, disorder, disease, or deficiency comprises type 1 diabetes or hypothyroidism. Suitably, the defect, disorder, disease, or deficiency comprises a deficiency of parathyroid hormone, a deficiency of an adrenal hormone, a deficiency of an ovarian hormone, a deficiency of a male gonadal hormone, a deficiency of a pituitary hormone, a deficiency of a thyroid hormone, a deficiency of a secreted factor from liver tissue, or a deficiency of a secreted factor from gut tissue. Suitably, the adrenal hormone may be adrenaline, aldosterone, androstenedione, cortisol, dehydroepiandrosterone, estrogen, or noradrenaline. Suitably, the ovarian hormone may be estradiol or progesterone. Suitably, the male gonadal hormone may be testosterone or androstenedione. Suitably, the pituitary hormone may be human growth hormone, adrenocorticotropic hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, prolactin, antidiuretic hormone, or oxytocin. Suitably, the thyroid hormone may be triiodothyronine or thyroxine. Suitably, the secreted factor from liver tissue may be insulin-like growth factor-1 , angiotensinogen, thrombopoetin, or hepcidin. Suitably, the secreted factor from gut tissue may be glucose-dependent insulinotropic peptide, glucagon-like peptide 1 , gastrin, secretin, or cholecystokinin. The above deficiencies are typically caused by decreased or absent production or secretion of the respective compounds due to defects in the producing (endocrine) cells. The deficiency will typically be determined by a physician, in the light of the target hormone or factor, the relevant circumstances, including the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. Suitably, the deficiency may be defined as a situation wherein the subject exhibits at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% lower mean amount or concentration of the hormone or factor compared to a control subject group, for example as measured in a sample obtained from the subject.

[0203] “Treatment” as used herein encompasses an increase of the amount or concentration of target hormone or factor, for example as measured in a sample obtained from the subject, of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% when compared to the amount or concentration of target hormone or factor before treatment.

[0204] Kits

[0205] In another aspect the invention provides a kit (of parts) for enriching or isolating cell clusters comprising ex vivo differentiated cells comprising: a. a first volume having a density in the range of from about 1 .0 g / ml to about 1 .3 g / ml; b. optionally, a second volume having a higher density compared to the highest density of the first volume; c. optionally, a third volume having a lower density compared to the lowest density of the first volume; and d. optionally, instructions for carrying out the method according to the invention. The first, second, and optional third volume, when combined form a density gradient medium. Suitably, the first volume may have a density of from about 1 .0 g / ml to about 1 .3 g / ml, of from about 1.0 g / ml to about 1.2 g / ml, of from about 1.0 g / ml to about 1.1 g / ml, of from about 1.02 g / ml to about 1.20 g / ml, of from about 1.02 g / ml to about 1.14 g / ml, of from about 1.03 g / ml to about 1.30 g / ml, of from about 1.03 g / ml to about 1.15 g / ml, of from about 1.04 g / ml to about 1.20 g / ml, of from about 1.04 g / ml to about 1.16 g / ml, of from about 1.04 g / ml to about 1.12 g / ml, of from about 1.05 g / ml to about 1.20 g / ml, of from about 1.05 g / ml to about 1.15 g / ml, of from about 1.05 g / ml to about 1.13 g / ml, of from about 1.05 g / ml to about 1.11 g / ml, of from about 1.05 g / ml to about 1.09 g / ml, of from about 1 .053 g / ml to about 1 .088 g / ml, of from about 1 .053 g / ml to about 1 .097 g / ml, of from about

[0206] 1 .053 g / ml to about 1 .081 g / ml, of from about 1 .060 g / ml to about 1 .075 g / ml, of from about 1 .060 g / ml to about 1 .080 g / ml, or of from about 1 .060 g / ml to about 1 .070 g / ml. Preferably, the first volume may have a density of from about 1.053 g / ml to about 1.088 g / ml.

[0207] Suitably, the second volume may have a density of more than about 1 .3 g / ml, more than about 1 .30 g / ml, more than about 1 .2 g / ml, more than about 1 .20 g / ml, more than about 1.16 g / ml, more than about 1.15 g / ml, more than about 1.14 g / ml, more than about 1.13 g / ml, more than about 1.12 g / ml, more than about 1.11 g / ml, more than about 1.1 g / ml, more than about 1.10 g / ml, more than about 1 .09 g / ml, more than about 1 .088 g / ml, more than about 1 .097 g / ml, more than about 1 .081 g / ml, more than about 1 .075 g / ml, more than about 1 .080 g / ml, or more than about 1 .070 g / ml.

[0208] Suitably, the third volume may have a density of less than about 1 .0 g / ml, less than about 1 .02 g / ml, less than about 1 .03 g / ml, less than about 1 .04 g / ml, less than about 1 .05 g / ml, less than about 1 .053 g / ml, or less than about 1 .060 g / ml.

[0209] Suitably, the first volume may have a single, discrete density, for example a density of 1 .09 g / ml. Suitably, the first volume may have multiple discrete densities, preferably contained in separate sub-volumes, for example a density of 1 .05 g / ml in a first sub-volume and a density of 1 .09 g / ml in a second sub-volume. Suitably, the first volume may comprise at least a first and a second subvolume. Suitably, the first sub-volume may have a density of about 1 .0 g / ml, about 1 .01 g / ml, about 1 .02 g / ml, about 1 .03 g / ml, about 1 .04 g / ml, about 1 .05 g / ml, about 1 .06 g / ml, about 1 .07 g / ml, about 1 .08 g / ml, about 1 .09 g / ml, or about 1.1 g / ml. Suitably, the first sub-volume may have a density of about 1 .040 g / ml, about 1 .045 g / ml, about 1 .047 g / ml, about 1 .048 g / ml, about 1 .049 g / ml, about 1 .050 g / ml, about 1 .051 g / ml, about 1 .052 g / ml, about 1 .053 g / ml, about 1 .054 g / ml, about 1 .055 g / ml, about 1 .056 g / ml, about 1 .057 g / ml, about 1 .058 g / ml, about 1 .059 g / ml, about 1 .060 g / ml, about 1 .061 g / ml, about 1 .062 g / ml, about 1 .063 g / ml, about 1 .064 g / ml, about 1 .065 g / ml, about 1 .066 g / ml, about 1 .067 g / ml, about 1 .068 g / ml, about 1 .069 g / ml, about 1 .070 g / ml, about 1 .071 g / ml, about 1 .072 g / ml, about 1 .073 g / ml, about 1 .074 g / ml, or about 1 .075 g / ml. Preferably, the first sub-volume may have a density of about 1 .05 g / ml. Preferably, the first sub-volume may have a density of about 1 .053 g / ml.

[0210] Suitably, the second sub-volume may have a density of about 1 .3 g / ml, about 1 .25 g / ml, about 1.2 g / ml, about 1.20 g / ml, about 1.19 g / ml, about 1.18 g / ml, about 1.17 g / ml, about 1.16 g / ml, about 1.15 g / ml, about 1.14 g / ml, about 1.13 g / ml , about 1.12 g / ml , about 1.11 g / ml, about 1.1 g / ml, about 1.10 g / ml, about 1.09 g / ml or about 1.08 g / ml. Suitably, the second sub-volume may have a density of about 1 .099 g / ml, about 1 .098 g / ml, about, 1 .097 g / ml, about 1 .096 g / ml, about 1 .095 g / ml, about 1 .094 g / ml, about 1 .093 g / ml, about 1 .092 g / ml, about 1 .091 g / ml, about 1 .090 g / ml, about 1 .089 g / ml, about 1 .088 g / ml, about 1 .087 g / ml, about 1 .086 g / ml, about 1 .085 g / ml, about 1 .084 g / ml, about 1 .083 g / ml, about 1 .082 g / ml, about 1 .080 g / ml, about 1 .079 g / ml, about 1 .078 g / ml, about 1.077 g / ml, about 1.076 g / ml, or about 1.075 g / ml. Preferably, the second sub-volume may have a density of about 1.09 g / ml. Preferably, the second sub-volume may have a density of about 1 .088 g / ml.

[0211] Suitably, the first volume may have a gradient of densities, for example a continuous density gradient, such as of from 1.1 g / ml to 1 .3 g / ml. Suitably, the first volume may have a gradient of densities that is within the range of from about 1 .0 g / ml to about 1 .3 g / ml, of from about 1 .0 g / ml to about 1.2 g / ml, of from about 1.0 g / ml to about 1.1 g / ml, of from about 1.02 g / ml to about 1.20 g / ml, of from about 1.02 g / ml to about 1.14 g / ml, of from about 1.03 g / ml to about 1.30 g / ml, of from about 1.03 g / ml to about 1.15 g / ml, of from about 1.04 g / ml to about 1.20 g / ml, of from about 1.04 g / ml to about 1.16 g / ml, of from about 1.04 g / ml to about 1.12 g / ml, of from about 1.05 g / ml to about 1.20 g / ml, of from about 1.05 g / ml to about 1.15 g / ml, of from about 1.05 g / ml to about 1.13 g / ml, of from about 1.05 g / ml to about 1.11 g / ml, of from about 1.05 g / ml to about 1.09 g / ml, of from about 1.053 g / ml to about 1 .088 g / ml, of from about 1 .053 g / ml to about 1 .097 g / ml, of from about 1 .053 g / ml to about 1 .081 g / ml, of from about 1 .060 g / ml to about 1 .075 g / ml, of from about 1 .060 g / ml to about 1 .080 g / ml, or of from about 1 .060 g / ml to about 1 .070 g / ml. Preferably, the first volume may have a gradient of densities that is within the range of from about 1 .053 g / ml to about 1 .088 g / ml.

[0212] General definitions

[0213] As used herein, the term “ex vivo” refers to “outside” the body. The term “in vitro” can be used to encompass “ex vivo” components, compositions and methods. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper

[0214] Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0215] As used herein, the term “substantially” refers to a great extent or degree. The exact allowable degree may depend on the specific context. For example, the term “substantially” in the context of an isolated or enriched cell clusters substantially consisting of a specific cell type as disclosed herein would mean that they may comprise at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%, of the specific cell type.

[0216] Examples

[0217] Aspects of the invention are demonstrated by the following non-limiting examples.

[0218] Example 1

[0219] MATERIALS AND METHODS

[0220] In vitro culture and differentiation of hPSCs.

[0221] Human embryonic stem cell line RC9 and HUES8 were obtained from Roslin institute and WiCell, respectively. The human induced pluripotent stem cell lines LUMC iPSC1 , LUMC iPSC2, LUMC iPSC3 (Lumc-Gmp-ipsc_donor-02 Line 1 / 2 / 3) was generated in the GMP facility of Leiden University Medical Center (LUMC) (37, 39)). HUES8 was cultured on laminin 521 (Biolamina, catalog no. LN521) and RC9 and LUMC iPSC1 were cultured on vitronectin (Thermo fisher, catalog no. A14700)-coated plates in Essential 8 (E8) medium (Thermo Fisher, catalog no. A1517001) and passaged using Versene (Gibco, catalog no. 15040-033). To prepare for the differentiation experiments, 70% confluent 2D culture flasks of hPSC were dissociated using Accutase (Stemcell Technologies, catalog no. 07920) and seeded in E8 supplemented with 10 pM Rho-Associated kinase inhibitor (ROCKi, Y-27632; Stemcell Technologies, catalog no. 72304) at a density of 0.5 million cells per ml in either 125 ml-disposable spinner flask (Corning; 3152) or 30ml-disposable bioreactor (ABLE® Biott®; ABBWVS03A-6) on a magnet stir plate set to 60 rpm in a 37 °C incubator, 5% CO2, and 100% humidity. To start the differentiations, the medium was changed to DO medium 48 h post-seeding. The differentiation was carried out using a seven-stage protocol according to the previously published protocol (9), culturing the cells in a 3D suspension culture system (disposable bioreactors) for the course of differentiation for about a month. ‘GMP compliant’ reagents for this Example are considered as those available as clinical-grade aiming for a process entirely animal component-free. Complete media formulations are available in the Tables 1-2. Importantly, all culture including expansion and differentiation media were kept antibiotic-free in order to comply with GMP regulations. Yet, experiments reported in this Example have been performed with research-grade reagents. Media refreshments were performed daily throughout stage 1 to 5 and every other day in stage 6 and 7.

[0222] Table 1 : Basal medium and SC-islet medium composition:

[0223] Table 2: Stage-specific media compositions:

[0224] Primary human islets.

[0225] Donor islets were obtained from cadaveric human organ donors. Human islet isolations were performed in our institute (2). Islets were used for research only if they could not be used for clinical purposes and if research consent was present, according to Dutch national laws. Islets were cultured in regular CMRL 1066 medium (5.5 mmol / L glucose), supplemented with 10% human serum, 10 mM HEPES (Lonza, BEBP17-737E), 2 mM L-glutamine (Lonza, BEBP17-605E), Nicotinamide 1.2mg / ml (Apotheek AZL, 97996807), 20 pg / ml ciprofloxacin (Fresenius Kabi, 15999149) and 50 pg / ml gentamycin (Centrafarm b.v., RVG 57572) in the Accessory Platelet Bags, (Terumo BCT, 70030) at 37°C in 5% CO2-humidified atmosphere and medium was refreshed the day after isolation and every two days thereafter.

[0226] Immunohistochemistry and image analysis.

[0227] Samples of in vitro cultures were fixed for 30 min and samples of explanted grafts were fixed overnight in 4% PFA and embedded in paraffin. Sections (4 pm) were deparaffinized and subjected to 0,01 M citrate buffer, pH 6.0, boil in autoclave. The slides were blocked with 5% normal goat serum, 0.3% triton-100 in PBS, and incubated with primary antibodies diluted in PBS / BSA 1 % / 0.3 triton overnight in +4 °C. Secondary antibodies were diluted similarly and incubated for 1 h at RT. Antibodies are listed below. The slides were imaged with Confocal SP8 WLL and Airyscan. Images were processed in LAS X (Leica Application Suite X) and ZEN 2012 software.

[0228] Primary antibodies used: rabbit anti-SYP (Monosan, cat no: PS340) dilution 1 : 100; mouse anti-CK19 (Cell Signaling, cat no: 4558) dilution 1 :200; rat anti-C-peptide (lgG2) (DSHB, cat no: GN-ID4) dilution 1 :200 and 1 :400; mouse anti-glucagon (IgG 1 ) (Sigma, cat no: G2654) dilution 1 : 100; mouse anti-Ki67 (Invitrogen, cat no: 180192Z) dilution 1 :200; mouse anti-NKX6.1 (lgG1) (DSHB, cat no: GN-1 D4) dilution 1 :100; rabbit anti-MAFA (IgG) (Bethyl Laboratories, cat no: A700-067) dilution 1 :100; rabbit anti-PDX1 (IgG) (Cell Signaling, cat no: 5679S (D59H3)) dilution 1 :100.

[0229] Secondary antibodies used: vs rabbit anti-SYP: goat anti-rabbit (IgG) AF647 (Invitrogen, cat no: A21244) dilution 1 :500; vs mouse anti-CK19: goat anti-mouse (IgG) AF568 (Thermo Fisher, cat no: A-11004) dilution 1 :500; vs rat anti-C-peptide: goat anti-rat (IgG) AF488 (Invitrogen, cat no: A11006) dilution 1 :500; vs mouse anti-glucagon, mouse anti-Ki67, and mouse anti-NKX6.1 : goat anti-mouse (IgG 1 ) AF568 (Invitrogen, cat no: A21124) dilution 1 :500; vs rabbit anti-MAFA and rabbit anti-PDX1 : goat anti-rabbit (IgG) AF568 (Invitrogen, cat no: A11011) dilution 1 :500.

[0230] Flow cytometry.

[0231] SC-islets were dispersed into a single-cell suspension with TryplExpress (Gibco, catalog no. 12604- 013) for 40 min at 37C. Dispersed SC-islets were filtered through cell strainer 40uM (Pluriselect, catalog no. 43-50040-51) and fixed with BD Cytofix fixation buffer (BD Biosciences, catalog no. 554722) at 4 °C for 20 min, washed twice in 1x BD Perm / Wash buffer (BD Biosciences, catalog no. 554722). Cells were incubated with perm / wash for 10 minutes for intracellular staining, at room temperature, counted the single cells by NC200 (Nucleocounter, Chemometec) and incubate 0.2 million cells with conjugate antibodies. After 2h, cells were washed with 1x BD Perm / Wash buffer and 1x with PBS / EDTA, then transferred to flow cytometry tubes (BD Falcon) and were analyzed for relevant stage-specific marker expression Table 3. using Aurora spectral flow cytometers (Cytek, Aurora 3 laser) and Spectroflo for the data analysis.

[0232] Table 3: Antibodies for flow cytometry:

[0233] Glucose-stimulated C-peptide secretion.

[0234] Static assays of C-peptide secretion were carried out in 1 .5 ml tubes. A total of 40 SC-islets were handpicked and incubated in Krebs buffer (KRB) with 1 .67 mM glucose for 90 min for equilibration, and then exposed to sequential 60-min incubations of 1 .67 mM, 20 mM, 1 .67 mM glucose, followed by 15min exposure to 30 mM KCI Krebs (depolarization challenge). Dynamic assays of C-peptide secretion were performed using a perfusion apparatus (Biorep v5) with a flow rate of 0.05 ml min-1, and sampling every 5 min. 25 handpicked SC-islets were exposed to 1 .67 mM glucose for 28 min, 20 mM glucose for 60 min, 1.67 mM glucose for 15 min and 30 mM KCL for 10 min. After the tests, the SC-islets were collected and the C-peptide and DNA contents were analyzed. C-peptide concentration was determined for supernatant and cell lysate samples using the Human Ultrasensitive C-peptide ELISA (Mercodia, catalog no. 10-1141-01). The DNA content was measured by Picogreen assay (Thermo Fisher, P7589). In some instances C-peptide secretion levels were normalized to total C-peptide content for each sample. In other instances C-peptide secretion levels and C-peptide content were normalized to the DNA content in each sample.

[0235] Stimulation indices were calculated as a ratio of C-peptide secretion at high glucose (20 mM) relative to the basal secretion (1 .67 mM glucose).

[0236] Seahorse assay.

[0237] Oxygen consumption rates (OCR) of SC-islets and donor islets were measured using a Seahorse Bioscience XFe96 Extracellular Flux Analyzer and analyzed on Agilent Wave software v.2.6. SC- islets and donor islets were loaded into Matrigel-coated Seahorse XF96 cell culture microplates (20 clusters per well) and attached to the surface overnight in 5mM-glucose SC-islet medium (Table 1). Before the assay, the medium was exchanged into KRB containing 1 mM glucose, and the islets allowed to equilibrate for 60-90 min at 37 °C. OCRs were measured over 156 min. Basal OCR was calculated before the sequential addition of a stimulatory nutrient (20 mM glucose), an inhibitor of ATP-synthetase activity (5 pM oligomycin), a mitochondrial uncoupling agent (4 pM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, FCCP) and finally an inhibitor of Complex I of the electron transport chain (1 pM / 1 pM antimycinA / rotenone) in KRB. Respiration rates were normalized to the basal OCR before nutrient or small molecule addition. To compare the absolute level of OCR between SC-islets and adult islets, raw OCR values were normalized to the total protein content of each well, measured by BCA assay (Thermo Fisher, 23225).

[0238] Transmission electron microscopy.

[0239] SC-islets were processed as previously described (41). Briefly, SC-islets were fixed in 1 .5% gutaraldehyde / 0,1 M cacodylatebuffer for 1 hour at room temperature for conventional transmission electron microscopy. SC-islets were kept in 1 % OsO4 / 1 .5% RuO4 / 0.1 M cacodylate buffer on ice for 1 hour. The SC-islets were collected in 0.2% Agar / MQ. The pellet with the SC-islets was dehydrated in a series of ethanol, and followed up by a series of epon (LX112, lead research):aceton mixtures. Each piece of the pellet was put at the bottom of a beem capsule and filled with EPON. The beem capsules were polymerized in a 70°C oven for 2 days. Ultrathin sections (90nm) were made using a Leica EM Ultracut 6 and contrasted with 7% uranylacetate (10 min) and Reynolds lead citrate (5 min). Sections were examined on a Fei Tecnai Twin transmission electron microscope (FEI, Eindhoven, Netherlands) at 120 kV, with a Gatan Oneview camera (Gatan, Pleasonton) on binning 2. Image analysis was performed using Aperio Imagescope (Leica). For immunogold labeling the SC-islets were fixed in 2% Paraformaldehyde( PFA) / 0,2% glutaraldehyde in 0,1 M PHEM buffer during 1 hour. The SC-islets were embedded in 10% gelatin / PBS. Then samples were cryoprotected in a 2,3M sucrose / PBS solution for 1 hour and frozen in liquid nitrogen. Ultrathin cryosections (90 nm) were made with a Leica EM Ultracryotome, then incubated with primary C-peptide antibody (DSHB, GN-ID4; 1 :16000) and secondary rabbit anti rat antibody (1 :200), followed by 10 nm Protein A gold particles (1 :300) in 1% BSA in PBS. The sections were embedded (methylcellulose, 0.3 % uranylacetate solution) and examined as described above.

[0240] Imaging of intracellular Ca2+ activity.

[0241] Stem cell clusters were loaded with Cal-520 AM (Abeam, ab171868) calcium indicator dye, dissolved in DMSO to a stock concentration of 2mM, and diluted to a final working solution of 5uM in HBSS (ThermoFisher, 14025092) containing 10 mM Hepes and 1 .6 mM glucose. Clusters were incubated for 60-90 minutes in an incubator with physiological conditions of 37 °C and 5% CO2, followed by an incubation of 30 minutes at room temperature, and a wash with HBSS (10 mM Hepes, 1 .6 mM glucose). Clusters were imaged in a 4 well p-slide (ibidi, 80426). Experiments were performed in 37 °C HBSS containing 10 mM Hepes. Samples were imaged using a commercially available DragonFly500 spinning disk system (Andor) on a DMi8 microscope (Leica) with a Plan Apo 10x objective. Microscope setup was controlled by Fusion software (Andor), and images were taken with a Zyla sCMOS camera (Andor). Images were aquired in low glucose (1 .67 mM) for 3 minutes, high glucose (20 mM) for 10 minutes, and KCI (30 mM) for 3 minutes, at 1 Hz. Analyses and image processing were performed with FIJI (Imaged), by determining a Region of Interest (ROI) for 5 cells, and measuring the mean grey value over time. Since Cal-520 is a single-wavelength calcium indicator, calculations were made for the recorded fluorescence divided by the initial fluorescence (F / FO). Visualization of data was performed in GraphPad Prism 6.0.

[0242] Single cell-RNA sequencing.

[0243] SC-islets were dispersed into a single-cell suspension with TryplExpress (Life Technologies) for 40min at 37C until a single-cell suspension was achieved. Dispersed SC-islets were filtered through cell strainer 20uM (Pluriselect, 43-50020-03) to remove cell clumps, counted the single cells by NucleoCounter® NC-200™ (Chemometec), then centrifuged at 200 g for 2 min. Single cells with the viability of more than 85% were frozen in Cryostor (Sigma, C2874) for further 10x scRNAseq analysis by 10X Genomics 3’ V3 chemistry protocol. The single cell-RNA sequencing was performed by Single Cell Discoveries according to the standard 10x Genomics protocols. Cryopreserved cells were thawed and counted prior to loading on the 10X Genomics controller. The resulting sequencing libraries were prepared following a standard 10x Genomics protocol. The sequencing results were mapped with Cell Ranger count (10X Genomics) using the human reference CRGh38-2020A. Every dataset generated was filtered and processed using the R-based workflow Seurat (version -4.3.0) (Fig.2 and 7) (42, 43). Cells containing between 200 and 10000 unique feature counts and less than 40%mitochondrial gene content were considered for down-stream analysis in Seurat. Datasets were normalized and log-transformed using a scaling factor of 10000. In order to identify the dimensionality in each dataset principal component analysis was assessed on the 2000 variable genes. Graph-based clustering was used to group cells using the Louvain algorithm to optimize standard modularity function. Then, Uniform Manifold Approximation and Projection (uMAP) was performed for dimensional reduction. The p cell clusters were identified in each dataset (HUES8 (n=2), RC9 (n=3)) based on the expression of insulin and other beta cell signature genes. Next, to perform the comparative analysis, the inventors extracted p cells and exclusively merged the clusters of SC-p cells and the pancreatic p cells from the primary human pancreatic dataset (n=3 donors; using the Gene Expression Omnibus (GEO) (https: / / www.ncbi.nlm.nih.gov / geo / ; GEO accession: GSE218316). Finally, the inventors obtained the average expression values for each desired gene using the Average Expression function in Seurat. Graphs were generated using the ggplot2 (49) and Seurat visualization R packages..

[0244] Regarding Fig. 4K-M, each dataset generated was filtered and processed using the Pythonbased Scanpy workflow (version 1 .9.1). Mean Absolution Deviation (MAD) was used to filter low- quality cells and cells with less than 10% mitochondrial gene content were considered for downstream analysis. Additionally, Kcnq1ot1 and Malatl were removed from the downstream analysis because they have been reported to be linked to mapping errors. Doublet removal was performed using scDblFinder (version 1 .16.0) and ambient RNA contamination was corrected using SoupX (version 1 .6.2). Datasets were normalized and log-transformed using a scaling factor of 10000. In order to identify the dimensionality in each dataset principal component analysis was assessed on the 2000 variable genes. Graph-based clustering was used to group cells using the Leiden algorithm, an improved version of the Louvain algorithm (56) to optimize the standard modularity function. Then, Uniform Manifold Approximation and Projection UMAP) was performed for dimensional reduction. The inventors used datasets of SC-islets before and after enrichment (HUES8 (n=1 )) to analyze the effect of the enrichment method. Datasets were merged using Scanpy. Clusters were annotated based on differential gene expression analysis of the normalized count matrix and the expression of key genes shown in Fig. 11 P (expression was scaled from 0 to 1 , based on maximum and minimum mean expression) and according to the annotation from earlier reports (10). Next, SC-p clusters were merged together with p cells from donor islets (n=3 donors; GEO accession: GSE218316) for further downstream analysis (Fig. 11 M-O).

[0245] Transplantation studies. Animal experiments were approved by the animal welfare committee of the Leiden University Medical Center (LUMC). Transplantations were performed in 5-12 week old male (immunodeficient) NSG-RIP-DTR mice (44). Briefly, stage-7 SC-islets were implanted under the kidney capsule of mice. Animals were anaesthetized with isoflurane inhalation. A small incision was made in the kidney capsule by using a 27G needle tip, and space was created between the kidney ant the capsule. Cells were collected in a cannula and transplanted underneath the capsule by using a Hamilton syringe. Body weight and non-fasted blood glucose values (from tail tip blood) were monitored twice weekly post-surgery. Functionality of SC-derived islets was tested by performing an intra-peritoneal glucose tolerance test (IPGTT) at 14, 28, 56, and 90 days post-surgery. In addition, mice engrafted with enriched SC-islets were assessed up to 170 days post transplantation. Briefly, blood samples (~100pL) were taken from the tail vein after 4 hours of fasting (t=0). Glucose was administered (2g / kg), and blood glucose values were tested at 15, 30, 60, and 120 minutes. Additional blood samples were taken at 30 and 60 minutes. Concentration of human c-peptide was tested on the blood plasma with Human Ultrasensitive C-peptide ELISA (Mercodia, catalog no. 10- 1141-01). At the end of the experiments, animals were sacrificed and organs were harvested and fixed in 4% PFA. Engrafted kidneys were further processed for histological analysis.

[0246] Dithizone Staining.

[0247] The zinc-chelating dye dithizone (DTZ) (Sigma, Cat No. D5130) stock solution was prepared with 50 mg of DTZ in 10 ml of dimethyl sulfoxide (DMSO) and equilibrium to 50 ml DPBS. The staining solution was filtered through a 0.45 pm nylon filter and then used as the DTZ working solution. The SC-islets were stained for 5 min in the DTZ solution. Then, SC-islets were rinsed three times with DPBS and imaged by light microscope (Olympus CKX53).

[0248] Density gradient separation method.

[0249] (Stage 7) SC-islets were transferred from a 3D culture system as described above in ‘In vitro culture and differentiation ofhPSCs’ for use in subsequent density gradient separation without using any cell dissociation methods or products (e.g. cell dissociation buffers or enzymes). SC-islets were cultured in islet medium with 5 mM glucose 24 h before purification. A density gradient with a continuous linear slope was formed in a 50 mL tube starting, starting at 1 .045 g / mL and ending at 1.100 g / mL by using a clinically-approved contrast agent (Xenetix) diluted in University of Wisconsin solution (UW) with the ratio calculated according to the Table 4, where the density of UW (Light solution) is 1.045 g / ml, the density of Xenetix 350 (Heavy solution) is 1.401 g / ml, and the density of 80.34% UW + 19.66% Xenetix is 1.115 g / ml. Before purification, SC-islets were soaked in UW solution for 30 min at 4 °C. Hereafter, cell clusters were gently loaded on top of the gradient. Then, SC-islets were centrifuged at 200 g for 7 min with the acceleration at 7 g and deceleration at 3 g at 4 °C. Seven fractions of 7 mL of gradient medium were collected and distributed across 50 ml tubes. Cell clusters from each fraction were collected by passing through cell strainer with the pore size of 0.45 pm and washed in islet medium to remove the gradient components (Xenetix and UW solutions). Then, the density of collected gradient medium was re-measured for validation by density meter (Anton Paar, DMA-35N). Subsequently, each fraction was cultured individually in SC-islet medium (CMRL1066 (Corning, 15-110-CVR) + 2 mmol / L GlutaMAX + 2% Alburex20 + 1 :200 ITS-X + 10 pg / mL heparin + 10 pmol / L zinc sulfate + 10 mM HEPES (Lonza, BEBP17-737E) + 1.2 mg / ml Nicotinamide (Apotheek AZL, 97996807) + 0.5 mM Sodium Pyruvate (Lonza, BE13-115E) + 1 :2000 Trace elements A (Corning, 25-021 -Cl) + 1 :2000 Trace elements B (Corning, 99-175-CI) + 1 :2000 Lipid concentrate (Gibco, 11905-031)) or stage 7 medium (Table 2) at 37 °C in 5% CO2 for 1d before evaluation. Fractions 2 to 5 were pooled for some further analysis, where mentioned as “Enriched SC-islet”. Fractions 1 , 6, and 7 were pooled and mentioned as “Depleted SC-islet”.

[0250] Table 4: Calculation of linear density gradient:

[0251] INTRODUCTION

[0252] The progressive loss of insulin-producing p cells is a hallmark of type 1 diabetes (T 1 D), a chronic autoimmune condition resulting from a combination of genetic and environmental factors. An insufficient functional p cell mass leads to an elevation of the blood sugar level (hyperglycemia), which can lead to ketoacidosis and death when left untreated. Transplantation of exogenous p cells to replace dead or dysfunctional endogenous p cells represents a promising functional cure allowing proper control of blood glucose levels in patients with T1 D. Human pluripotent stem cells, including both embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC), constitute a virtually unlimited source of cells for islet replacement therapy. In an attempt to develop an off-the-shelf supply of p cells for transplantation, several multi-stage protocols have been developed to convert pluripotent stem cells into stem cell-derived islets (SC-islets) containing p cells in vitro, with recent advances leading to the generation of glucose-responsive insulin-secreting cells in a full 2D, 2D / 3D (7-9) or full 3D culture system (10). Full 3D culture systems are more suitable for manufacturing large amounts of SC-islets for clinical application. Also, the next generation of protocols will need to be fully defined and entirely animal component-free in order to strictly meet the Good Manufacturing Practice (GMP) standards required for the manufacturing of cell-based product. Clinical studies have started using an earlier stage cell product (pancreatic endoderm) (13) and later stage cell product (stem cell islets) (14). Unwanted heterogeneity of the final cell product defined as the presence of non-target cells after directed differentiation is one of the main challenges in the regenerative medicine field regarding the in vitro production of any cell type. It is currently impossible to avoid the generation of non-target, non-islet cells throughout the differentiation process, partly due to a limited knowledge on human islet developmental biology. While the majority of cells follow the intended path at every stage of the process, others deviate off. As long as non-target cells are present in the final cell product, implementing a strategy to enrich for clusters enriched for the target stem cell-derived islets (SC- islets) as the last step of the manufacturing process is relevant from a translational point of view, increasing efficiency while lowering the transplant volume, resulting in the generation of a potential safer cell product.

[0253] To date, enrichment methods mainly rely on the use of specific cell surface markers for antibodybased sorting of subpopulations of interest, which requires the dissociation of the SC-islets to single cells in order to specifically sort the target population. These procedures will cause major cell loss and raise the risk of cellular stress in the remaining cells in addition an increased risk of contamination due to the additional manipulations. Furthermore, all these strategies will disrupt the specific organization between the different islet cell types present in the SC-islets, while the spatial arrangement and communication by paracrine signaling are known to significantly influence p cell function (30-33). Therefore, developing a purification method which maintains the cytoarchitecture and composition of SC-islets will be beneficial for preservation of p cell survival and function. Herein, a full 3D suspension, easily scalable and clinically compliant method is presented that includes an enrichment step for target cell clusters using a density gradient separation as a novel method to manufacture SC-islets for clinical application.

[0254] RESULTS

[0255] SC-islets are heterogeneous in cell composition for target endocrine cells and non-target, non-islet cells.

[0256] In order to develop a scalable, full 3D suspension and GMP compliant protocol for clinical application, a 7-stage (~30-day) differentiation protocol was devised, based on earlier protocols (8- 10), and applied the standards of GMP manufacturing to provide a GMP-compliant differentiation protocol (Fig. 6A). Cells were cultured (and differentiated) in antibiotics-free conditions to adhere to GMP regulations. Also earlier reports indicated that the presence of antibiotics could affect the differentiation efficiency. Furthermore, cells were cultured in xeno-free conditions to keep the process animal-component free. No BSA (bovine serum albumin) or Matrigel were used. BSA was replaced by human albumin fraction IV (Alburex).

[0257] The protocol was optimized using the embryonic stem cell lines HUES8 (35) (research-grade) and RC9 (clinical-grade) (36) as well as clinical-grade iPSC lines (37, 39). Stage 7 cell products (‘S7 SC- islets’) consist of endocrine cells rich (dithizone staining positive) clusters and endocrine cells poor clusters (Fig 1A). S7 SC-islets generated from HUES8 and RC9 cell lines contained on average 52% SC-p cells (C-peptide expressing cells), 8% SC-a cells (glucagon expressing cells), and 4% of SC- islet cells were positive for both C-peptide and glucagon (Fig. 1 B and Fig. 1C). Following an expansion of the number of experiments, the following results were obtained: S7 SC-islets generated from HUES8 and RC9 cell lines contained on average 56.4 ± 5.0 % SC-p cells (C-peptide expressing cells), 8.5 ± 1.3 % SC-a cells (glucagon expressing cells), 5.5 ± 0.9 % of SC-islet cells were positive for both C-peptide and glucagon, and 21.1 ± 4.9 % of the cells were double positive for C-pep and NKX6.1 (Fig. 1J). The SC-p cells (C-peptide expressing cells) showed expression of NKX6.1 , PDX1 , and NEUROD1 and only rare cells were positive for the MAFA (P cell maturity marker) (Fig. 1C). The differentiation efficiency was comparable in 3 iPSC lines (Fig. 6C). In addition, the inventors found 8.6 ± 1.3 % of somatostatin-positive cells, and 16.5 ± 4.2 % SC-EC (Enterochromaffin: SLC18A1 expressing cells), as non-islet endocrine cells (Fig. 6D-E).

[0258] Ultrastructure analysis by electron microscopy revealed that SC-p cells display insulin granules, 200- 300 nm in diameter, with an electron dense core surrounded by a less dense halo, indicating some level of maturity. Immunogold labeling for C-peptide validated the presence of insulin granules within SC-p cells at the ultrastructural level (Fig. 1 D).

[0259] SC-is / ets show limited glucose-induced C-peptide secretory capacity.

[0260] A key feature of mature p cells is their capacity to respond to a glucose challenge by increasing insulin (or its by-product C-peptide) secretion, which returns back to a basal secretion level upon subsequent exposure to low glucose. S7 SC-islets generated from HUES8 and RC9 were evaluated, in parallel to primary human donor islets obtained through the inventor’s human islet isolation facility. The total human C-peptide content in S7 SC-islets was 179.1 ± 42 (ng) / DNA (pg) (HUES8, n=11), 25.6 ±14 (ng) / DNA (pg) (RC9, n=9), 899.5 ± 173.1 (ng) / DNA (pg) (donor islets, n=10) (Fig. 1 E and 1 K). Basal C-peptide concentrations were similar compared to primary human islets. Upon glucose stimulation, S7 SC-islets from both stem cell lines secreted 4% of the total C-peptide content in response to a glucose challenge, as compared to 16% of the total C-peptide content in donor islets (Fig. 1 F and 1 L). The stimulation index [ratio of stimulated C-peptide to basal C-peptide] of SC-islets was 1 .2 ± 0.5 (HUES8) and 1 .6 ± 1 .2 (RC9), while donor islets treated in parallel showed a stimulation index of 4.5 ± 3.1 (Fig. 1 G and 1 M).. Of note, the stimulation index of primary human donor islets is highly variable between islet cell preparations from different donors, depending on a variety of circumstances including donor characteristics, ischemia time, culture duration, and aspects related to the isolation procedure as was previously reported. Finally, human C-peptide secretion from SC-p cells returned properly to a basal secretion level when the cells were subjected to low glucose again (Fig. 1 F). In a dynamic perfusion system the SC-p cells showed a stimulation index of 2.2 ± 0.1 in the first 5 minutes after exposure to high glucose (Fig. 6B). Upon exposure to KCI, which depolarizes the cell membrane, a 15.8 ± 9.5 fold increase in C-peptide concentration was observed as compared to baseline (Fig. 6B). Following an expansion of the number of experiments, the following results were obtained: in a dynamic perfusion system the SC-p cells showed a statistically significant (p=0.031) response to glucose with a stimulation index of 1 .9 ± 0.5 in the first 5 minutes after exposure to high glucose (Fig. 6F). Upon exposure to KCI, which depolarizes the cell membrane, a 12.6 ± 7.3 fold increase in C-peptide concentration was observed as compared to baseline (Fig. 6F).

[0261] Functional adult islets are also characterized by increased mitochondrial respiration upon a glucose stimulus, which is associated with glucose-stimulated insulin secretion, and indicates metabolic coupling to insulin release. An increase in oxygen consumption rate (OCR) was found that is glucose-dependent in primary donor islets but variable and often absent responses in S7 SC-islet batches (Fig. 1 H and I). The maximal respiratory capacity and spare capacity were also reduced in SC-islets of both cell lines as compared to primary islets, indicating a lesser ability of SC-islet cells to produce ATP at their maximum rate and a reduced response to increased energy demand and stress..

[0262] Altogether, SC-islets generated by this GMP compliant, full 3D differentiation protocol presented an organotypic cytoarchitecture and composition similar to primary donor islets. The cells showed reduced glucose responsiveness and glucose-dependent oxygen consumption. The cells showed insulin secretory capacity, though at a reduced level compared to primary islets.

[0263] SC- / 3 cells displays a similar transcriptional signature as primary donor islet-fi cells.

[0264] In order to obtain an in-depth characterization of SC-p cells, single-cell RNA sequencing was performed to compare their transcriptional profile to the one of p cells from primary human islets (‘donor islet-p cells’). The inventors first assessed the expression of the selected p cell identity genes as defined by a previous study (38). It was found that, despite some differences between the two cell lines, HUES8-P and RC9-P cells shared similarity in p cell identity and functionality gene expression profile with the donor islet-p cells, including INS, PDX1 , NKX6.1 , SLC30A8, ABCC8, PCSK1 , ENO1 , ENOI B, UCHL1 , NEUROD1 , NKX2.2, CDKN1C among others (Fig. 2A and Fig. 7). In addition, SC-p cells expressed genes involved in p cell maturation, metabolic sensing and signaling, function and secretion (CANA1C, SLC30A8, GRN, PTPRN, CAMK2N1), exocytosis (VAMP2, CDC42, STX1A, SNAP25, STXBP1), glucose sensing (ABCC8, KCNJ11 , SLC2A2) (Fig. 2B and E). Also, SLC25A1 , which is responsible for transporting citrate across the inner mitochondrial membrane and a key step in the TCA cycle (Fig. 2B), had a similar expression as donor-p cells. However, SC-p cells displayed lower expression of some other genes associated with P cell maturation (UCN3, MAFA), metabolic sensing and signaling (ADCYAP1 , VGF), function and secretion (KCNK3, G6PC2) (Fig. 2B). Disallowed genes such as HK1 , HK2, SLC16A1 were downregulated in SC-p cells, though not to the same extent as in donor islet cells (Fig. 2B). Of note, low expression of the so-called disallowed genes is essential for proper metabolic sensing and control over glycolytic flux, leading to the upregulation of glycolysis and TCA cycle genes (Fig. 2C). Finally, genes associated with respiration in mitochondria, the electron transport chain complexes including MT-ND3 (Complex I), SDHB (Complex II), UQCRFS1 (Complex III), MT-CO1 (Complex IV) and the MT-ATP6 (Complex V) showed similar expression pattern in SC-p cells compared to donor islet-p cells, indicative of relatively functional mitochondria in SC-p (Fig. 2D; Fig. 7A, B), which is consistent with the mitochondrial oxygen consumption profile (Fig. 1 G and H). Further analysis of hallmark genes related to oxidative phosphorylation and protein secretion, using the MSigDB database, confirmed the similarity between donor-p cells and SC-p in many of the relevant gene expression (Fig. 2, Fig. 7B and C).

[0265] Overall, it was shown that the differentiation protocol, also when applied to the clinical-grade RC9 cell line, generated insulin-producing cells with a transcriptional (identity and metabolism related) profile comparable to primary human donor islet-p cells.

[0266] SC / 3 cells function in vivo after transplantation.

[0267] In order to assess the capacity of SC-islets to function in vivo, cell clusters generated from both cell lines were transplanted under the kidney capsule of immunodeficient mice and the mice were subjected to a glucose challenge at different time points post-transplantation (Fig. 3A). Stimulated human C-peptide was detectable from 14 days post-transplantation already, indicating successful engraftment of the S7 SC-islets. Furthermore, the inventors observed an increase in stimulated human C-peptide secretion over time, both in HUES8-SC-islet and RC9-SC-islet transplants (Fig. 3C and D). Human C-peptide secretion increased with a fold change of 12.6 and 11 .9 on day 90 compared to day 14 in HUE8- and RC9-islet cell transplants, respectively (Fig. 8A to 8D). Following an expansion of the number of experiments, the following results were obtained: the inventors observed an increase in stimulated human C-peptide secretion over time, both in HUES8-SC-islet and RC9-SC-islet transplants. Human C-peptide secretion increased with a fold change of 8.2 and 7.3 on day 90 compared to day 14 in HUE8- and RC9-islet cell transplants, respectively (Fig. 3E and F). Furthermore, the inventors observed a significant (p=0.02) increase in human C-peptide released at 30’ post glucose injection compared to the basal secretion from day 90 post-transplantation onward (Fig. 10).

[0268] Finally, the graft-bearing kidney was removed at the end of the 3 month experiments. Immunostaining validated the presence of SC-islets containing a majority of C-peptide positive p cells, and a fraction of glucagon expressing a cells (Fig. 3B). Of note, only rare bihormonal cells (coexpressing both C-peptide and Glucagon) were found (data not shown).

[0269] In summary, SC-islets generated from this protocol engrafted well in vivo and showed evidence of functional maturation of human SC-p cells over time. This highlights the potential of SC-islets as surrogate cells for the development of cell-based replacement therapies when applying a GMP- compliant protocol.

[0270] Enrichment of SC-islets by a clinically applicable density gradient separation.

[0271] Next, it was reasoned that the differentiation protocol could be further improved by adding a final step to enrich the final cell product for cell clusters rich in (islet) target cells in order to generate a product of lower volume, potentially better and more consistent efficacy. In density gradient separation, also called isopycnic, buoyant or equilibrium separation, cell clusters are separated on the basis of their density. It was hypothesized that endocrine cell-rich clusters would have a different density from the (non-target) endocrine cell-poor clusters and could therefore be separated from each other. A linear (continuous) density gradient was established using clinically applicable reagents (UW solution, commonly used for organ preservation, and Xenetix, a CT contrast agent, as heavy component) ranging in density between 1 .045 and 1.100 g / ml (Fig. 4A and 4B and Table 4). The gradient was validated by assessing the density of each fraction using a density meter (Fig. 4B). Following centrifugation, the gradient was divided in 7 fractions of similar (gradient medium) volume. The tissue enriched in each of the fractions was subsequently cultured in islet medium. Importantly, it was found that cell viability was not affected as assessed up to 4 days and up to 10 days post density gradient separation (Fig. 4C).

[0272] The inventors stained SC-islets with dithizone (which stains insulin-containing cells) in order to assess the purity of each fraction (Fig. 4D and 9A), and quantified the amount of tissue obtained per fraction (Fig. 9B). The normalized purity of each fraction showed that fractions 2 to 5 contained most SC-islets (Fig 4E). These findings were confirmed by total C-peptide content (Fig 9D). For the subsequent experiments, fractions 2 to 5 were pooled together in a so-called ‘enriched SC-islet’ fraction that comprised of 93.9 ± 0.8 % DTZ+ tissue (Fig. 4F). Normalized C-peptide content indicated that enriched SC-islets contained 73.8 ± 3.8 % C-peptide (Fig. 9E). Overall, enriched SC- islets (pooled fractions 2, 3, 4, 5) displayed a two-fold higher C-peptide content as compared to depleted SC-islets (fractions 1 , 6 and 7 pooled together) (Fig. 9D and 9E).

[0273] The amount of tissue collected in the enriched SC-islet fraction contained 78 ± 13% of the initial tissue mass, indicating that the tissue volume for transplantation was reduced by 22 ± 13% (Fig. 9B and 9C). Of note, enriched SC-islets were mostly present at a density of 1 .053-1 .088 g / ml.

[0274] Following an expansion of the number of experiments, the following results were obtained: the inventors stained SC-islets with dithizone in order to assess the purity of each fraction (Fig. 4D and 11A), and quantified the amount of tissue obtained per fraction (Fig. 11 B). The normalized purity of each fraction showed that fractions 2 to 5 contained most SC-islets (Fig 4I). These findings were confirmed by total C-peptide content (Fig 11 C). For the subsequent experiments, fractions 2 to 5 were pooled together in a so-called ‘enriched SC-islet’ fraction that comprised of 92.8 ± 1.5 % DTZ+ tissue (Fig. 4J). Normalized C-peptide content indicated that enriched SC-islets contained 67.6 ± 3.6 % C-peptide (Fig. 11 D). A similar range of purity is obtained when assessed based on the frequency of DTZ+ clusters in the enriched fractions compared to non-purified cells (Fig. 11 E). Overall, enriched SC-islets (pooled fractions 2, 3, 4, 5) displayed a two-fold higher C-peptide content as compared to depleted SC-islets (fractions 1 , 6 and 7 pooled together) (Fig. 11C and D), which is consistent across cell lines (RC9, HUES8, LUMC iPSC1) (Fig. 11 F).

[0275] The amount of tissue collected in the enriched SC-islet fraction contained 82.2 ± 3.6 % of the initial tissue mass, indicating that the tissue volume for transplantation was reduced by 17.8 ± 3.6% (Fig. 11 G). Of note, enriched SC-islets were mostly present at a density of 1 .053-1 .088 g / ml.

[0276] Further characterization by immunostaining confirmed the presence of islet cell rich (synaptophysin- (SYP, endocrine) and C-peptide (C-pep) and glucagon (GCG) positive) clusters in enriched SC- islets, whereas the depleted SC-islet fraction showed an enrichment in duct cell (CK19) positive clusters (Fig. 4G, 11 H, and 111). Transmission electron microscopy and immunogold labeling validated the presence of insulin granules in cells from enriched SC-islets (Fig. 4H).

[0277] Finally, the inventors performed single-cell transcriptomics showing the identity of the cell types present in the preparation before vs after enrichment (Fig. 4 l-K and Fig. 11 P-Q). These include islet cells, exocrine cells, neuro-endocrine cells (GAP43+ NE) but also off-target cell populations such as enterochromaffin cells and mesenchymal cells, in line with findings from others. Importantly, this data confirmed an enrichment in endocrine cells from 54% to 80% post-density gradient separation, and a strong reduction in non-endocrine cells (early ductal, ductal, mesenchymal, neuroendocrine) from 29% to 2% post-density gradient separation. Of note, the expression of identity and functionality genes in SC-p cells remained stable after enrichment (Fig. 11 J-L). Also, genes associated with respiration in mitochondria, the electron transport chain complexes including MT- ND3 (Complex I), SDHB (Complex II), UQCRFS1 (Complex III), MT-CO1 (Complex IV), MT-ATP6 (Complex V) and CYCS (Cytochrome c) remained unaffected (Fig. 11 M-O).

[0278] Enriched SC-islets are functional in vitro and in vivo.

[0279] Next the functionality of the purified cell clusters was assessed. The stimulation index during glucose-stimulated insulin secretion of the enriched SC-islets was increased (p = 0.043) as compared to non-purified preparations (S7 SC-islets) (Fig. 5A). Following an expansion of the number of experiments, the following results were obtained: the functionality of the purified cell clusters was assessed by dynamic glucose-stimulated C-peptide secretion test. SC-islets and enriched SC-islets showed a comparable stimulation index after exposure to high glucose level (Fig. 5F). Upon exposure to KCI, which depolarizes the cell membrane, enriched SC-islets showed a 26.1 ±18.6 fold increase in C-peptide concentration, which was 2 times higher as compared to SC-islets before enrichment (12.8± 5.9) (Fig. 5G). This reflects the higher C-peptide content as also shown in Fig. 5H. Mitochondrial function was unaltered after the purification procedure (Fig. 5B). Notably, enriched SC-islets showed an increased glucose-induced calcium activity upon exposure to high glucose as compared to non-purified cell clusters, while showing a low and stable intra-islet calcium activity ([Ca2+]i) at low glucose (Fig. 5C).

[0280] Finally, enriched SC-islets engrafted well in vivo (Fig. 5D) and showed an increased stimulated human C-peptide secretion compared to non-purified S7 SC-islets (Fig. 5E). A longer follow-up indicated that the enriched SC-islets were functional up to 6 months post-transplantation (Fig. 5I and 12A-D), Interestingly, mice transplanted with SC-islets and enriched SC-islets, and in particular enriched SC-islets compared to SC-isled engrafted for a longer period of time (60 d or more), showed a more rapid glucose clearance over time (Fig 12H-K), and displayed lower non-fasting blood glucose levels reaching the human glycemic setpoint (~5 mM) from 3 months onward (Fig 12G), altogether indicating improved functionality of the engrafted SC-p cells. Yet, importantly the inventors never observed any case of severe hypoglycemia with a follow-up up to 6 months posttransplantation, indicating that the secretion of C-peptide is regulated. Histological analyses at the time of transplantation (Fig. 12E) and after 170 days (Fig. 5J) confirmed the enrichment in synaptophysin (SYP, endocrine), C-peptide (C-pep) and glucagon (GCG)-positive cells in the graft, and the reduced frequency of CK19 (ductal) cells and associated cystic structures (Fig. 5J). Of note, enterochromaffin cells remained present in grafts, also post-enrichment (Fig. 12L). Also, it was observed that the expression of the p cell maturity marker MAFA was enhanced posttransplantation, providing additional evidence for further maturation of the graft (Fig. 12F). Importantly, the density gradient separation was successfully performed in two hESC lines HUES8 and RC9 and three GMP-hiPSC lines (Fig. 4E, F, I, and J, Fig. 5A, B, E-H, Fig. 9, and Fig. 11A-G), demonstrating the robustness of the separation method.

[0281] Altogether these data validated density gradient separation as a GMP-compliant purification method to enrich for SC-islet preparations for endocrine cell-rich clusters in the final cell product, while showing no adverse effect on the biological activity of the cells.

[0282] DISCUSSION

[0283] Here, a GMP-compliant, full 3D suspension, differentiation protocol with enrichment in SC-islets with enrichment in endocrine cell-rich clusters by density gradient separation for clinical application is presented.

[0284] SC-p cells resulting from this protocol displayed key genes associated with p cell identity and function. Yet, in accordance with the literature, these cells mostly lack the expression of maturity genes such as MAFA and showed limited glucose-dependent insulin secretion and oxygen consumption. Nevertheless, SC-islets can engraft properly and mature further after transplantation. Despite the major progress in the differentiation protocols, one of the main challenges for manufacturing hPSC-based therapies remains to get rid of non-target cells in order to ensure a (reduced) transplant volume, safety, and efficacy. It was found that the full 3D suspension differentiation protocol as presented here generated endocrine cell-rich clusters and endocrine cellpoor clusters. The inventors developed a GMP-compliant purification technique relying on the principle of density gradient separation. This method enabled the inventors to enrich for SC-islet preparations in endocrine cell-rich clusters based on the density of the cell cluster, regardless of its size. Cell cluster size only influences the rate at which cell clusters move until their density is the same as the surrounding gradient medium.

[0285] This method enabled enrichment in endocrine cells and depletion of non-endocrine cells leading to a more consistent cell product with reduced non-target cells. The viability and functionality of the cells were unaffected as the procedure does not disturb the cell cluster cytoarchitecture and cell-cell contacts that are well known to play a critical role in the maintenance of p cell health and function in the context of primary islets. Importantly, purification of SC-islets by this method allows to deplete the cell preparation in non-target cells without disturbing the cytoarchitecture and cell-cell contacts that are well known to play a critical role in the maintenance of p cell health and function in the context of primary islets (30-33). This is in contrast with all the previously reported purification methods, which rely on the dissociation of the cell product to single cells, combined to an antibodybased detection of the target cell type by FACS or MACS technologies. The enrichment procedure did not impair the viability and functionality of the cells. In addition, the functionality of SC-islets including C-peptide secretion and intra-islet calcium activity is improved after enrichment. Importantly, this density gradient separation method is easily scalable to large volumes of cultures, and at reasonable costs. Further optimization of gradient conditions will be needed to narrow the SC-islet fractions to a more limited number of fractions only, while widening the distance between islet cell rich and non-target cell clusters leading to a better separation and reduced SC-islet loss. Furthermore, although it is envisioned that the procedure can be adapted to other cell products than SC-islets, it can be foreseen that cell type specific optimization of the gradient components (and density range) will be required.

[0286] Overall, here density gradient separation is proposed as a robust, easily scalable, and clinically applicable technique for effective purification of SC-islets. Enrichment of the endocrine populations in the final cell product is expected to improve transplantation efficacy and safety.

[0287] References

[0288] 2. M. F. Nijhoff et al., Glycemic Stability Through Islet-After-Kidney Transplantation Using an Alemtuzumab-Based Induction Regimen and Long-Term Triple-Maintenance Immunosuppression. Am J Transplant 16, 246-253 (2016).

[0289] 7. D. Balboa et al., Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nat Biotechnol 40, 1042-1055 (2022).

[0290] 8. V. Gorgogietas et al., GLP-1 R agonists demonstrate potential to treat Wolfram syndrome in human preclinical models. Diabetologia, (2023).

[0291] 9. F. Fantuzzi et al., In depth functional characterization of human induced pluripotent stem cell- derived beta cells in vitro and in vivo. Front Cell Dev Biol 10, 967765 (2022).

[0292] 10. A. Veres et al., Charting cellular identity during human in vitro beta-cell differentiation. Nature 569, 368-373 (2019).

[0293] 13. A. M. J. Shapiro et al., Insulin expression and C-peptide in type 1 diabetes subjects implanted with stem cell-derived pancreatic endoderm cells in an encapsulation device. Cell Rep Med 2, 100466 (2021).

[0294] 14. T. W. REICHMAN et al., 836-P: Glucose-Dependent Insulin Production and Insulin- Independence in Type 1 Diabetes from Stem Cell-Derived, Fully Differentiated Islet Cells — Updated Data from the VX-880 Clinical Trial. Diabetes 72, (2023).

[0295] 30. F. C. Wieland, M. M. J. P. E. Sthijns, T. Geuens, C. A. van Blitterswijk, V. L. S. LaPointe, The Role of Alpha Cells in the Self-Assembly of Bioengineered Islets. Tissue Eng Pt A 27, 1055-1063 (2021).

[0296] 31 . Y. H. Jo et al., Artificial Islets From Hybrid Spheroids of Three Pancreatic Cell Lines. Transpl P46, 1156-1160 (2014). 32. C. Kelly, H. G. Parke, J. T. McCluskey, P. R. Flatt, N. H. McClenaghan, The role of glucagon and somatostatin-secreting cells in the regulation of insulin release and beta-cell function in heterotypic pseudoislets. Diabetes-Metab Res 26, 525-533 (2010).

[0297] 33. C. A. v. B. Fredrik C. Wieland, Aart van Apeldoorn, Vanessa L.S. LaPointe, The functional importance of the cellular and extracellular composition of the islets of Langerhans. Journal of Immunology and Regenerative Medicine, (2021).

[0298] 35. C. A. Cowan et al., Derivation of embryonic stem-cell lines from human blastocysts. New Engl J Med 350, 1353-1356 (2004).

[0299] 36. P. A. De Sousa et al., Derivation of the clinical grade human embryonic stem cell line RCeO13-A (RC-9). Stem Cell Res 17, 36-41 (2016).

[0300] 37. Novoa, J. J., et al., Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products. Cytotherapy, 2024. 26(6): p. 556-566.

[0301] 38. L. van Gurp et al., Generation of human islet cell type-specific identity genesets. Nat Commun 13, 2020 (2022).

[0302] 39. Novoa, J., et al., Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system. Cytotherapy, 2024.

[0303] 41 . H. S. Spijker et al., Conversion of mature human beta-cells into glucagon-producing alpha cells. Diabetes 62, 2471-2480 (2013).

[0304] 42. L. van der Maaten, G. Hinton, Visualizing Data using t-SNE. J Mach Learn Res 9, 2579-2605 (2008).

[0305] 43. R. Satija, J. A. Farrell, D. Gennert, A. F. Schier, A. Regev, Spatial reconstruction of single-cell gene expression data. Nat Biotechnol 33, 495-U206 (2015).

[0306] 44. K. Furuyama et al., Diabetes relief in mice by glucose-sensing insulin-secreting human a-cells. Nature 567(7746), 43-48 (2019).

[0307] 49. Wickham, H., ggplot2. Wiley Interdisciplinary Reviews-Computational Statistics, 2011. 3(2): p. 180-185.

[0308] 56. V. A. Traag, L. Waltman, N. J. van Eck, From Louvain to Leiden: guaranteeing well-connected communities. Sci Rep 9, 5233 (2019); published online EpubMar 26 (10.1038 / s41598-019- 41695-z).

[0309] Example 2

[0310] MATERIALS AND METHODS

[0311] For this example, the same materials and methods were generally used as set out for Example 1 , unless otherwise indicated.

[0312] Density gradient separation

[0313] Human embryonic stem cell lines RC9 or HUES8 or human induced pluripotent stem cell lines were ex vivo differentiated to Stage 7 SC-islets according to Example 1 and were enriched using a discontinuous gradient. The Stage 7 SC-islets were transferred from a 3D culture system for use in subsequent density gradient separation without using any cell dissociation methods or products (e.g. cell dissociation buffers or enzymes). The discontinuous gradient was created with Xenetix 350 (Guerbet, p = 1.401) as heavy component and was mixed with university of Wisconsin solution (UW, p = 1.045 g / cm3, osmolality = 320 mOsm / kg) to achieve the desired density solution. A discontinuous gradient was prepared by creating four working solutions with a density of 1.050 g / cm3; 1.065 g / cm3; 1.075 g / cm3and 1.090 g / cm3, as calculated in Table 5. A 50 mL falcon tube was loaded with the gradient by gently top-layering it manually at a 45° angle starting with a density of 1.090 g / mL and ending with 1 .050 g / cm3, creating a 4-segmented discontinuous gradient. Before loading the SC-islets, the cells were incubated in UW-solution for 30 minutes at 4°C. Subsequently, 7 mL of the cell suspension was loaded gently on top of the gradient. Then the SC-islets were centrifuged at 200 g for 7 minutes, with an acceleration of 7 g and a deceleration of 3 g, at 4°C. The cells of each fraction were collected by passing through cell strainer with a pore size of 0.45 pm and remove the gradient components by washing them with CMRL medium. To validate the quality of the density gradient, the density of each filtered fraction was remeasured using a density meter (Anton Paar, DMA 35 basic). Finally, the cells of each fraction were cultured individually at 37°C in 5% CO2 for 24 hours before evaluation. Each fraction was evaluated individually. Additionally fraction 3 and 4 were pooled as enriched SC-islets and fractions 1 , 2 and 5 were pooled as depleted SC-islets.

[0314] Table 5: Calculation of discontinuous density gradient:

[0315] RESULTS

[0316] This study was designed based on the continuous density gradient separation protocol as set out in Example 1 . However, here a discontinuous gradient was used for density gradient separation instead. In a discontinuous density gradient setting, It was shown that the purest fractions were fractions 3 and 4 which correspond to a density range of 1 .063-1 .072 g / cm3.

[0317] In order to correct for the turbulence at the bottom of the tube and to mitigate the target-cell loss, the density of fraction 5 was adjusted to 1 .090 g / cm3. The adjusted gradient design was subsequently used to compose the same gradient using UWS as light component, with Xenetix as heavy component. The experiment was repeated three times, and the densities were measured with a density meter to validate for their density after centrifugation. The heavy gradient component yielded comparable experimental density values after centrifugation, with only slight deviations from the calculated target density. Five distinct fractions and four interfaces were generated. Compared to the continuous gradient design of Example 1 , an additional interface appeared between fraction 4 and 5. This interface was not discernible previously, as mixing occurred in the bottom of the tube, resulting in a lower density after centrifugation. As expected, the density of fraction 5 is lower than intended; however, a distinct interface is observable, which mitigates the migration of target cells to fraction 5.

[0318] The inventors conducted an Ultrasensitive C-peptide ELISA and a PicoGreen assay to compare the distribution of C-peptide positive cell clusters and other cells. The individual fractions were analyzed, as well as pooled enriched and depleted fractions. The experiments were repeated three times for each gradient component. It was observed that a discontinuous gradient controlled with Xenetix significantly enriched C-peptide-positive cells in the enriched fractions (fraction 3 and 4). When comparing the cell distribution in the enriched and depleted fractions, it can be observed that more than 60% of the SC-islets were collected in the enriched fractions (fraction 3 and 4). Less than 40% of the remaining tissue was collected in the depleted fractions (fraction 1 , 2 and 5) (Figure 13B). However, the data suggest that this tissue is not rich in C-peptide (Figure 13A). When comparing the individual fractions for their C-peptide content following density gradient separation, the highest amount of C-peptide positive cells was collected in fraction 3 and 4 (Figure 13E). A large proportion of the targeted cells migrated to fraction 2 (26.2%), which was not rich in C-peptide (Figure 13, E and F).

[0319] In conclusion, Example 2 established that a discontinuous density gradient separation according to the present invention can be used for enriching or isolating C-peptide-positive cell clusters derived from iPSC.

Claims

Claims1 . A method of enriching cell clusters comprising ex vivo differentiated cells, the method comprising: a. subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and b. isolating the cell clusters from the first volume, thereby enriching the cell clusters.

2. A method of isolating cell clusters comprising ex vivo differentiated cells, the method comprising: a. subjecting a suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and b. isolating the cell clusters from the first volume.

3. The method according to claim 1 or 2, wherein the first volume has a density of from about 1 .0 g / ml to about 1 .3 g / ml, or wherein the first volume has a gradient of densities that is within the range of from about 1 .0 g / ml to about 1 .3 g / ml.

4. The method according to any one of claims 1 to 3, wherein the first volume has a density of from about 1 .053 g / ml to about 1 .088 g / ml, or wherein the first volume has a gradient of densities that is within the range of from about 1 .053 g / ml to about 1 .088 g / ml.

5. The method according to any one of claims 1 to 4, wherein the density gradient medium or the first volume has a continuous, linear, discontinuous, segmented, or isokinetic density gradient.

6. The method according to any one of claims 1 to 5, wherein the ex vivo differentiated cells comprise or substantially consist of cells derived from stem cells, preferably cells derived from pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), or mesenchymal stem cells.

7. The method according to any one of claims 1 to 6, wherein the ex vivo differentiated cells comprise or substantially consist of cells derived from iPSC.

8. The method according to any one of claims 1 to 7, wherein the ex vivo differentiated cells comprise endocrine cells, preferably thyroid follicular cells, thyroid parafollicular cells, parathyroid chief cells, parathyroid oxyphil cells, or enteroendocrine cells.

9. The method according to any one of claims 1 to 8, wherein the ex vivo differentiated cells comprise pancreatic enteroendocrine cells or pancreatic islet cells, preferably alpha cells and / or beta cells.

10. The method according to any one of claims 1 to 9, wherein the cell clusters, preferably the enriched or isolated cell clusters, are organotypic cell clusters, preferably wherein the organotypic cell clusters are organotypic pancreatic islets, organotypic thyroid follicles, or organotypic parathyroid follicles.11 . The method according to any one of claims 1 to 10, wherein the cell clusters, preferably the enriched or isolated cell clusters, are organotypic pancreatic islets.

12. The method according to any one of claims 1 to 11 , wherein the cell clusters, preferably the enriched or isolated cell clusters, have a mean diameter of at least about 20 pm, or of from about 20 pm to about 6 mm.

13. The method according to any one of claims 1 to 12, wherein the cell clusters, preferably the enriched or isolated cell clusters, each have of at least about 20 cells, or of from about 20 to about 10000 cells.

14. The method according to any one of claims 1 to 13, wherein the enriched or isolated cell clusters each comprise at least 50% C-peptide or insulin secreting cells of the total cell number of the respective cell cluster and / or at least 5% glucagon secreting cells of the total cell number of the respective cell cluster.

15. The method according to any one of claims 1 to 14, wherein the enriched or isolated cell clusters comprise at least 50% C-peptide or insulin secreting cells and / or at least 5% glucagon secreting cells.

16. The method according to any one of claims 1 to 15, wherein the enriched or isolated cell clusters each comprise at least 70% thyroid hormone secreting cells, preferably triiodothyronine (T3) and / or thyroxine (T4) secreting cells, of the total cell number of the respective cell cluster and / or at least 5% calcitonin secreting cells of the total cell number of the respective cell cluster.

17. The method according to any one of claims 1 to 16, wherein the enriched or isolated cell clusters comprise at least 70% thyroid hormone secreting cells, preferably triiodothyronine (T3) and / or thyroxine (T4) secreting cells, and / or at least 5% calcitonin secreting cells.

18. The method according to any one of claims 1 to 17, wherein the enriched or isolated cell clusters have a purity of at least 70%, preferably wherein the enriched or isolated cell clusters comprise at least 70% dithizone (DTZ) positive cells of the total number of enriched or isolated cells.

19. A pharmaceutical composition comprising a therapeutically effective amount of the enriched or isolated cell clusters obtained by the method according to any one of claims 1 to 18, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

20. Enriched or isolated cell clusters obtained by the method according to any one of claims 1 to 18, or the pharmaceutical composition according to claim 19, for use as a medicament.21 . Enriched or isolated cell clusters obtained by the method according to any one of claims 1 to 18 or the pharmaceutical composition according to claim 19, for use in the treatment of a defect, disorder, disease, or deficiency of endocrine cells, preferably wherein the defect, disorder, disease, or deficiency is type 1 diabetes or hypothyroidism.

22. A method of treating a subject that suffers from a defect, disorder, disease, or deficiency, comprising obtaining enriched or isolated cell clusters according to the method according to any one of claims 1 to 18, or the pharmaceutical composition according to claim 19, and administering to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition.

23. A method of treating a subject that suffers from a defect, disorder, disease, or deficiency of endocrine cells, comprising obtaining enriched or isolated cell clusters according to the method according to any one of claims 1 to 18, or the pharmaceutical composition according to claim 19, and administering to the subject a therapeutically effective amount of the enriched or isolated cell clusters or the pharmaceutical composition, preferably wherein the defect, disorder, disease, or deficiency is type 1 diabetes or hypothyroidism.

24. A kit for enriching or isolating cell clusters comprising ex vivo differentiated cells comprising: a. a first volume having a density in the range of from about 1 .0 g / ml to about 1 .3 g / ml; b. optionally, a second volume having a higher density compared to the highest density of the first volume; c. optionally, a third volume having a lower density compared to the lowest density of the first volume; and d. optionally, instructions for carrying out the method according to the invention.

25. A method of enriching cell clusters comprising ex vivo differentiated cells, the method comprising: a. culturing the cell clusters in a three-dimensional (3D) cell culture; b. obtaining a suspension comprising the cell clusters; c. subjecting the suspension comprising the cell clusters to density gradient separation in a density gradient medium comprising at least a first and second volume, wherein the first and second volumes have a different density; and d. isolating the cell clusters from the first volume, thereby enriching the cell clusters.

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