Low-dose x-ray irradiation for the elimination of unwanted cell growth in pluripotent stem cell-derived islet-like clusters

Abstract

The invention provides methods of irradiating PSC-derived islet-like clusters with low-dose X- ray; irradiated PSC-derived islet-like clusters for cell therapy with improved safety profile and / or reduced proliferative off-target cells; and irradiated PSC-derived islet-like clusters and cell transplants for use in treatment.

Description

[0001] Low-dose X-ray irradiation for the elimination of unwanted cell growth in pluripotent stem cell-derived Islet-Like Clusters

[0002] FIELD OF THE INVENTION

[0003] The invention provides methods of irradiating PSC-derived islet-like clusters with low-dose X- ray; irradiated PSC-derived islet-like clusters for cell therapy with improved safety profile and / or reduced proliferative off-target cells and / or reduced radiation-induced damage; and irradiated PSC-derived islet-like clusters and cell transplants for use in treatment.

[0004] BACKGROUND OF THE INVENTION

[0005] Type 1 diabetes (T1D) is a subtype of diabetes mellitus that is characterized by insulin deficiency. T1D is an autoimmune disease that leads to progressive destruction of insulinproducing beta cells of the pancreatic islet. Manifestations of T1D are the sudden onset of severe hyperglycemia and a rapid progression to diabetic ketoacidosis. The condition is lifethreatening unless treated with exogenous insulin. Advances in insulin therapies, such as the development of insulin analogues and novel approaches for the administration of insulin with pump systems and improved devices for blood glucose monitoring have all contributed to better treatment of T1D patients. Nevertheless, the majority of T1D patients do not achieve the glycemic targets set by national and international guidelines. The frequency of hypoglycemic episodes tends to increase in direct relation to the intensity of insulin therapy and, in extreme cases, can lead to coma and death.

[0006] Average life expectancy of T1D patients is about 10-15 years lower compared to the unaffected population, which is strongly linked to long-term and acute diabetic complications . Insulin therapy cannot replicate the real-time glucose sensing and finely tuned insulin secretory response of pancreatic beta cells, resulting in imperfect glycemic control and the associated long-term complications. Also, the avoidance of hypoglycemia is one of the largest unmet needs. The quality of life of T1D patients is severely affected due to the need for constant monitoring of glycemia levels, type and amount of food ingested and factors that may modify insulin response and glucose regulation such as infections or physical activity. Fear of acute and long-term complications as well as complications themselves also have a strong negative impact. Furthermore, long-term patient adherence is a challenge, especially in adolescent patients. Insufficient and / or inappropriate insulin secretion is also seen in patients with type 2 diabetes (T2D). T2D is a progressive disease driven by an interplay of increasing insulin resistance, metabolic derangement such as failure to appropriately suppress hepatic glucose production, and declining beta cell secretory responses. Physical inactivity, high body mass and genetic predisposition are important factors in the etiology of T2D. At advanced stages, patients need to switch from interventions such as dietary or behavioral changes, oral antidiabetic drugs and / or injectable GLP-1 analogs to insulin therapy. Insulin therapy in patients with T2D is associated with similar long-term and acute complications as in patients with T1D. There are also other conditions linked to insulin insufficiency and the need to intervene with insulin therapy, such as diabetes due to recurrent pancreatitis (also called T3D), pancreatectomy e.g. due to recurrent pancreatitis or pancreatic tumors, or forms of diabetes caused by genetic mutations. Diabetes types associated with a need for insulin therapy can also be categorized as severe autoimmune diabetes (SAID), severe insulin-deficient diabetes (SIDD), or severe insulin-resistant diabetes (SIRD).

[0007] The Diabetes Control and Complications Trial (DCCT) trial has shown that residual beta cell mass (indicated by stimulated C-peptide levels) in patients with diabetes undergoing insulin therapy is inversely correlated with both long-term complications and hypoglycemic events (Steffes et al. 2003), a finding that has been confirmed repeatedly since then (e.g. Gubitosi- Klug et al. 2021; Nathan 2021). Treatment options which can increase functional beta cell mass in patients with T1D or other forms of insulin-dependent diabetes are therefore expected to deliver clinical benefits to the patients by reducing long-term complications and hypoglycemic episodes. Restoring beta cell mass above the threshold needed for full glycemic control can enable full insulin independence, resulting in maximal patient benefit.

[0008] The transplantation of cadaveric human islets is a clinically validated option to restore beta cell mass in patients with diabetes (Rickels et al. 2019), and serves as proof-of-concept for beta cell replacement. Insufficient donor islet supply, side effects of immunosuppression (both on overall patient health and long-term beta cell function in transplanted islets) and limitations of immunosuppression leading to the destruction of beta-cells over time are limiting factors preventing the wide-ranging application of this treatment to all patients with diabetes.

[0009] Pluripotent stem cell (PSC)-derived islet-like cell clusters (ILCs) containing a high fraction of insulin-producing beta cells can be obtained by directed in vitro differentiation (Nostro et al. 2012; Pagliuca et al. 2014; Jennings et al. 2015; Rezania et al. 2014) and can overcome the key challenges of cadaveric islet transplantation, especially the limitations of donor islet availability, and substantial batch-to-batch variability of isolated donor islets, thereby offering an attractive therapeutic option for managing type 1 diabetes, and insulin-dependent diabetes in general.

[0010] Differentiation of ILCs in vitro recapitulates key stages of islet formation during embryonic development (Hogrebe et al. 2023). Minimizing the fraction of cells that make unwanted cell fate decisions at the various developmental decision points during differentiation (and thereby reducing off-target cell content), and maximizing the fraction of cells that fully complete endocrine islet cell fate commitment (including insulin-producing, NKX6.1 -positive mono- hormonal beta cells) are key objectives of process development for manufacturing of ILCs for cell therapy. However, obtaining cell populations containing, for example, close to 100% beta and other islet endocrine cells, and which are devoid of residual pancreatic precursor cells is challenging.

[0011] While human pancreatic endocrine cells (including PSC-derived cells) generally show very low levels of proliferation and are able to function over extended periods of time with very limited turnover especially once they have completed maturation (Meier et al. 2008), unwanted cell proliferation can occur in implanted PSC-derived ILC grafts, which can lead to a substantial increase in graft volume over time. While this typically does not affect the antidiabetic activity of beta cells contained in the graft, it is not acceptable in a clinical context (Augsornworawat et al. 2020; Chandra et al. 2022; Hiyoshi et al. 2024).

[0012] Unwanted cell growth sometimes occurred despite an overall high quality (high beta cell and endocrine fraction) flow cytometry profile of the implanted cells. Two kinds of unwanted cell growth / cell types were observed: a) ductal cysts and exocrine precursors and b) mesenchymal cells. A third category, residual pluripotent cells derived from the pluripotent starting material, are in general also of concern, due to their ability to form teratomas.

[0013] Due to the nature of the stepwise differentiation process simulating embryonic development, which must pass through a pancreatic progenitor stage on the way to endocrine / beta cells, residual non-endocrine cells are an almost unavoidable component of ILCs generated from PSCs, and therefore the occurrence of mesenchymal outgrowth appears to be a universally relevant problem in PSC-based diabetes cell therapy. Furthermore, detecting such small quantities of undifferentiated cells within a transplant is a major obstacle to PSC-derived ILCs.

[0014] Overall, there is a strong need for methods that are rapid, efficient, and comprehensive (= addressing most or all unwanted proliferative off-target cell types) while sparing desired therapeutic cell types (e.g. beta cells) and without adversely affecting ILC maturation or function, to provide PSC-derived ILC products for diabetes cell therapy which are fully functional, safe, and do not give rise to unwanted outgrowth of either ductal cysts and pancreatic progenitors, mesenchymal and other PDX1 -negative non-pancreatic cells, and / or teratomas.

[0015] Interventions should be rapid (not unduly prolong the manufacturing process) and should not induce additional process steps (e.g. due to washing / required washout of compounds) to lower manufacturing process cost and lower overall process risk (e.g. through contamination).

[0016] Interventions should also not induce safety problems de novo, like a senescent and fibrosisinducing phenotype, a pro-inflammatory phenotype adversely affecting graft function and / or survival, or DNA damage posing a safety risk, e.g. from alkylating or mutagenic compounds, or excessive irradiation doses.

[0017] They should also not induce significant reduction in process yield, and / or result in inacceptable adverse effects on cell composition.

[0018] OBJECTIVES AND SUMMARY OF THE INVENTION

[0019] The inventors have surprisingly found that they could reduce and / or eliminate residual proliferative unwanted cells by treating PSC-derived islet-like clusters with low-dose X-ray irradiation in vitro. They have found that a low-dose X-ray irradiation was sufficient to produce PSC-derived ILCs that did not form any ductal cysts, mesenchymal cell masses or pluripotent cell-derived cell masses (teratomas) after implantation. Furthermore, maturation and function of P-cells was unaffected by the irradiation. The low-dose X-ray irradiation did not induce any long-term DNA damage and did not induce senescence markers, such as SERPINE1 / PALI, MMP1, CXCL1 and MMP3. Despite the absence of long-term DNA damage, low-dose X-ray irradiation was found to induce a long-lasting suppression of proliferation. It also did not induce innate inflammatory cytokine expression (e.g. IFNB1, IFNG, CXCL8). Low-dose X-ray irradiation was also found to have no adverse effects on cryopreserved endocrine stage ILCs, and to be, due to the short irradiation times, highly compatible with integration into GMP manufacturing. Furthermore, using X-rays is feasible with normal laboratory or clinical equipment and therefore reduces the need for hazardous and expensive equipment, such as radioactive radiation sources.

[0020] As an underlying principle, the inventors aimed to eliminate the proliferating unwanted cell types or their precursors, while leaving the largely non-proliferating endocrine and especially beta cells unharmed.

[0021] X-rays can be considered a superior irradiation source compared to y-rays, beta-rays, alpha particles, protons, ions, or other alternatives, since they can be administered with relatively simple equipment suitable for integration in normal laboratories and GMP manufacturing sites, and do not require radioactive components.

[0022] X-ray irradiation can be applied while cells are outside of bioreactors or other culture vessels but inside closed containers, e.g. when they have been harvested for cryopreservation or shipping. It also can be applied to intact cell clusters without a dissociation and re-aggregation step, meaning that besides a short residence time in an irradiation device, no additional process, harvesting or handling steps need to be introduced.

[0023] In a first aspect, the invention provides a method of reducing and / or eliminating unwanted cell growth in PSC-derived islet-like clusters (ILCs) by low-dose X-ray irradiation in vitro, comprising a) obtaining a population of cells comprising PSC-derived ILCs and proliferative off-target cells; and b) irradiating the population of cells with low-dose X-ray, wherein the low-dose is less than 10 Gy.

[0024] Unwanted cell growth in PSC-derived ILCs can occur when unwanted proliferative cells, i.e. off-target cells, are present in the ILCs. Proliferative off-target cells can occur when a fraction of cells make unwanted cell fate decisions at various developmental decision points, and therefore do not fully complete endocrine islet cell fate differentiation. Such proliferative off- target cells may be ductal cells, which form pancreatic ductal cysts, and proliferating multipotent progenitors, i.e. exocrine precursors. These may arise from pancreatic progenitors cells present in the graft material that had, at the time of implantation, not yet committed to an endocrine fate. Ductal cells in grafts may also derive from cells acquiring a ductal fate already during differentiation.

[0025] A second source of proliferative off-target cells are graft-derived mesenchymal, non pancreatic cell masses. Mesenchymal cells may arise through conversion of non-endocrine pancreatic progenitor cells after implantation.

[0026] A third source of proliferative off-target cells are residual pluripotent cells. These are cells that are left-over from the source material of pluripotent cells that were used to generate the PSC- derived ILCs. Already small numbers of cell clusters containing pockets of pluripotent cells may cause teratoma formation over time.

[0027] Although this list is not exhaustive and should not be considered limiting, these three sources of unwanted proliferative off-target cells have been identified as the most clinically relevant.

[0028] Hence, in one embodiment, the unwanted cell growth arises from proliferative off-target cells selected from the group consisting of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells.

[0029] In a second aspect, the invention also provides an in vitro method of producing PSC-derived ILCs with improved safety profile suitable for cell therapy, the method comprising a) producing ILCs by culturing PSC under conditions suitable for the formation of ILCs; and b) irradiating the ILCs produced in step a) with low-dose X-ray, wherein the low-dose is less than 10 Gy.

[0030] As stated above, unwanted proliferative off-target cells can lead to ductal cell masses (ductal cysts), expanding mesenchymal masses, and teratomas, posing a safety risk to patients receiving PSC-derived ILC transplants. The method of the invention uses low-dose X-ray irradiation, eliminating and / or greatly reducing proliferative off-target cells in PSC-derived ILCs before transplantation, thereby improving the clinical safety profile of the ILC transplant. The term cell therapy in this regard refers to the transplantation of PSC-derived ILCs into patients. The inventors were surprisingly able to show that X-ray doses of less than 10 Gy were sufficient to eliminate unwanted cell growth arising from proliferative off-target cells, without negatively impacting maturation of function of PSC-derived ILC transplants.

[0031] In one embodiment, low-dose X-ray irradiation is performed on ILCs in which endocrine differentiation and cell fate selection has been completed to a significant extent, is close to fully completed, or fully completed. In one embodiment, low-dose X-ray irradiation is performed on ILCs between days 20 and day 28 after the start of in vitro differentiation. In another embodiment, low-dose X-ray irradiation is performed on ILCs on days 21, 22, 23, 24 or 25 after the initiation of in vitro differentiation. In one embodiment, low-dose X-ray irradiation is performed on ILCs between days 22 and day 24 after the start of in vitro differentiation. In a preferred embodiment, low-dose X-ray irradiation is performed on ILCs on day 23 of differentiation.

[0032] In one embodiment, the method further comprises a step of culturing the population of PSC- derived ILCs after low-dose X-ray irradiation for several days until the desired level of endocrine differentiation and / or beta cell maturation has been achieved. In one embodiment, the step of culturing the population of PSC-derived ILCs after low-dose X-ray irradiation is for 1 to 10 days. In one embodiment, the step of culturing the population of PSC-derived ILCs after low-dose X-ray irradiation is for 5 to 10 days. In one embodiment, the step of culturing the population of PSC-derived ILCs after low-dose X-ray irradiation is for 6 to 8 days. In a preferred embodiment, the step of culturing the population of PSC-derived ILCs after low-dose X-ray irradiation is for 7 days

[0033] In one embodiment, the method further comprises a step of monitoring whether the low-dose X-ray irradiated ILCs are free of proliferative off-target cells.

[0034] In one embodiment, monitoring whether the irradiated ILCs are free of proliferative off-target cells comprises determining the expression of one or more proliferation markers, preferably wherein cells positive for one or more proliferation marker are reduced compared to controls that were not irradiated.

[0035] In one embodiment, the one or more proliferation markers are cell cycle regulators or proteins phosphorylated during the cell cycle. In one embodiment, the one or more proliferation markers are selected from the group consisting of CDK1, pRB (phosphorylated retinoblastoma protein) MKI67, PCNA, and T0P2A. Proliferation can also be detected by quantifying cells with increased DNA content while in S-phase.

[0036] In another embodiment, monitoring whether the irradiated ILCs are free of proliferative off- target cells comprises detection of proliferative cells. In one embodiment, detection of proliferative cells is performed using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, ductal cyst formation and mesenchymal cell detection assay, or transplantation into immunodeficient animals.

[0037] Residual pluripotent cells can be detected in vitro by plating ILCs on a matrix and in media suitable for the growth of pluripotent cells. Detection occurs by quantifying the formation of colonies expressing typical pluripotent cell markers (e.g. alkaline phosphatase, OCT4, NANOG or TRA1-60) after a suitable time of culture, typically over several days.

[0038] Ductal cyst forming cells and / or the presence of residual pancreatic progenitors can be detected by embedding intact / non-dissociated ILCs in suitable matrices (e.g. Matrigel, or synthetic replacements) and culture over several days in growth supporting media. Ductal cyst size and thereby detection can be enhanced by adding modulators of cAMP (forskolin, IBMX) during end stages of culture. Visual scoring or suitable automated image analysis systems can be used to quantify cyst formation. qPCR and / or immunostaining can provide additional information on cystic and other growth observed. For example, the increase of proliferation marker expression or ductal epithelial marker expression (e.g. hKRT19, CFTR) can be monitored during incubation, and compared between conditions at the end of the assay.

[0039] Detection of mesenchymal cell growth can be accomplished by analysing ILCs cultured in the same way as for the detection of ductal cyst formation, by harvesting cells and quantifying suitable mesenchymal cell markers by qPCR. Suitable markers include COL6A2, CCDC80 and C0L1A1.

[0040] Irradiation by low-dose X-rays may not necessarily result in complete elimination of the unwanted cell types, but can also result in their replicative arrest. Therefore, low-dose X-ray irradiation can result in the arrest of unwanted cell growth post transplantation, although some unwanted cells may be still detectable prior to transplantation by sensitive methods. Therefore, key parameters for the assessment of successful suppression of unwanted cell growth from ILCs include the absence of outgrowth in suitable in vitro assays (such as plating assay to detect residual pluripotent cells, or ductal cyst formation and mesenchymal growth detection assay) and / or the absence of unwanted growth in mid- to long-term in vivo assessments such as transplantation of ILC material into suitable sites in immunodeficient mice.

[0041] In another aspect, the invention also provides a population of PSC-derived islet-like clusters (ILCs) suitable for cell therapy, obtained by irradiation with low-dose X-ray at a dose of less than 10 Gy in vitro.

[0042] In another aspect, the invention also provides a population of PSC-derived ILCs suitable for cell therapy obtained by any one of the methods of the invention.

[0043] In another aspect, the invention also provides a cell transplant comprising the population of PSC-derived ILCs of the invention.

[0044] In another aspect, the invention provides a cell transplant comprising a population of PSC- derived ILCs, wherein the PSC-derived ILCs were irradiated with low-dose X-ray at a dose of less than 10 Gy in vitro before forming the cell transplant.

[0045] In another aspect, the invention also provides a pharmaceutical composition comprising the population of PSC-derived islet-like clusters (ILCs) of the invention or the cell transplant of the invention and one or more pharmaceutically acceptable excipients.

[0046] In another aspect, the invention provides the population of PSC-derived ILCs of the invention, the cell transplant of the invention or the pharmaceutical composition of the invention for use in the treatment.

[0047] In another aspect, the invention provides the population of PSC-derived ILCs of the invention, the cell transplant of the invention or the pharmaceutical composition of the invention for use in the treatment of diabetes. In one embodiment, the treatment comprises transplantation of the in vitro irradiated PSC- derived ILCs in a subject suffering from diabetes. In one embodiment, the diabetes is selected from type I diabetes, type II diabetes and insulin-dependent diabetes. In one embodiment, the diabetes is type I diabetes. In another embodiment, the diabetes is type II diabetes. In yet another embodiment, the diabetes is a form of insulin-dependent diabetes caused by recurrent pancreatitis, pancreatectomy, or genetic mutations.

[0048] In another aspect, the invention also provides a population of PSC-derived islet-like clusters (ILCs) for use in treatment, wherein the ILCs have been in vitro irradiated with low-dose X-ray at a dose of less than 10 Gy prior to treatment, preferably equal or less than 4 Gy.

[0049] In yet another aspect, the invention also provides a population of PSC-derived islet-like clusters (ILCs) for use in the treatment of diabetes, wherein the ILCs have been in vitro irradiated with low-dose X-ray at a dose of less than 10 Gy prior to treatment, preferably equal or less than 4 Gy.

[0050] In another aspect, the invention also provides a cell transplant comprising PSC-derived isletlike clusters (ILCs) for use in the treatment of diabetes, wherein the ILCs have been in vitro irradiated with low-dose X-ray at a dose of less than 10 Gy prior to treatment, preferably equal or less than 4 Gy.

[0051] ILCs can be transplanted into several sites within the body of patients, including into the liver via portal vein infusion, intramuscularly, into adipose tissue, below the anterior rectus or other abdominal muscle sheath layers, subcutaneously, or near or within a pocket formed from the omentum. For extra-hepatic sites, transplantation can occur either in one step, or after prior generation of a pre-vascularized pocket. Transplantation can be done with or without the use of encapsulation devices, which can be open (non-immune-isolating, in some cases allowing ingrowth of capillaries into ILC grafts) or closed (immune-isolating).

[0052] In another embodiment, the ILCs irradiated with low-dose X-rays are monitored after implantation with suitable non-invasive imaging methods, such as fluorescence imaging, PET, SPECT, BLI, MRI, MPI, and / or ultrasonography.

[0053] In one embodiment, the low-dose X-ray irradiation does not induce (i) long-term DNA damage;

[0054] (ii) expression of innate immunity markers including CXCL8, interferon secretion and signalling,

[0055] (iii) expression of senescence markers; and / or

[0056] (iv) impairment of ILC cell composition, cell maturation or function.

[0057] In one embodiment, long-term DNA damage is measured by determining the expression of pH2AX. The term long-term DNA damage refers to DNA damage that is not transient.

[0058] In one embodiment, senescence markers are selected from the group consisting of SERPINE1 / PAI-I, MMP1, CXCL1 and MMP3. Preferably, in one embodiment, the low-dose X-ray irradiation does not induce expression of SERPINE1 / PAI-1.

[0059] In one embodiment, innate immunity markers are selected from the group of ISG15 CXCL10, IFNB1, IFNG and CXCL8. In one embodiment, innate immunity markers are selected from the group of IFNB1, IFNG and CXCL8.

[0060] In one embodiment, ILC cell maturation is determined by determining the levels of one or more hormone markers selected from the group consisting of C-peptide, glucagon (GCG), somatostatin (SST) and serotonin (5-HT) and / or one or more pancreatic cell markers selected from the group consisting of PDX1 (pancreatic and duodenal homeobox 1) and NKX6.1 (NK6 homeobox 1). In one embodiment, pancreatic cells are PDXl-positive and NKX6.1 -positive. In another embodiment, mono-hormonal beta cells are C-peptide / NKX6.1 double positive.

[0061] In one embodiment, ILC cell function is determined by measuring KCl-induced insulin secretion in vitro., and / or by measuring overall cell yield; and / or by measuring percentage of mono-hormonal beta-cells; compared to controls that were not irradiated.

[0062] In another embodiment, ILC cell function and maturation is assessed after transplantation into an animal or a patient, e.g. by determining the amount of secreted C-peptide per transplanted beta cell, and or the response to physiological and / or pharmacological stimuli like glucose bolus or mixed meal stimulation, response to GLP-1 or stabilized analogues thereof, or by determining the response to insulin secretagogues or depolarization, e.g. with arginine. In one embodiment, low-dose X-ray irradiation supresses the unwanted growth of ductal cysts, mesenchymal cell masses and / or residual pluripotent cell masses in the ILCs. Preferably, the unwanted growth occurs after transplantation of the ILCs.

[0063] In one embodiment, the ILCs comprise a decreased percentage of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells; after low-dose X-ray irradiation compared to controls that were not irradiated.

[0064] In one embodiment, the decreased percentage is at least 10%. In a preferred embodiment, the decreased percentage is at least 50%.

[0065] In one embodiment, some or all of the unwanted proliferative cells are arrested in proliferative capacity after low-dose X-ray irradiation.

[0066] In one embodiment, after low-dose X-ray irradiation the percentage of residual non-endocrine mitotic cells in the PSC-derived ILCs is less than 10%, preferably less than 1%.

[0067] In one embodiment, after low-dose X-ray irradiation, the low-dose X-ray irradiated ILCs of the invention form ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses at lower rates than controls that were not irradiated.

[0068] In one embodiment, after low-dose X-ray irradiation, the ILCs of the invention do not form detectable ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using an in vitro assay selected from the group consisting of cell plating assay, ductal and mesenchymal cell assay.

[0069] In one embodiment, the ILCs do not form detectable ductal cysts, mesenchymal cell masses and / or pluripotent cell masses for at least 5 years after implantation; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using non-invasive imaging, palpation, other clinical signs indicating spaceconsuming cell growth, changes in diagnostic circulating blood parameters, and / or histology.

[0070] In one embodiment, the ILCs do not form detectable ductal cysts, mesenchymal cell masses and / or pluripotent cell masses for at least 1 year after implantation; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using non-invasive imaging, palpation, other clinical signs indicating spaceconsuming cell growth, changes in diagnostic circulating blood parameters, and / or histology.

[0071] PSC-derived ILCs can be prepared from induced pluripotent stem cells (iPSC), such as human iPSCs, or from embryonic stem cells, such as human embryonic stem cells. PSCs may be derived by nuclear transfer.

[0072] In one embodiment, the PSC is from mammalian origin. In a preferred embodiment, the PSC is from human origin.

[0073] In one embodiment, the PSC is selected from induced PSC (iPSC) and embryonic stem cell (ESC). In a preferred embodiment, the PSC is an iPSC.

[0074] Hence, in a especially preferred embodiment, the PSC of the invention is a human iPSC.

[0075] FIGURE LEGENDS

[0076] Figure 1: Imaging (A) and cell count (B) after iPSC clusters were cultured in 3D for 6 days after exposure to increasing X-Ray irradiation doses. Cell count was performed after cluster dissociation. Example data from X-Ray exp. 7

[0077] Figure 2: Microscopy images of plated iPS cells imaged 4 days after irradiation and culture in 2D. Staining against ALPL to assess pluripotency. Later analysis not possible due to overgrowth of control sample and complete death of 0.8 Gy sample. Data from X-Ray Experiment 5.

[0078] Figure 3: Flow cytometry assessment of cell composition at day 22 in the control condition (no iPSC spike-in) vs. 5% pluripotent iPSC spike-in (no X-Ray) and 5% pluripotent iPSC spike-in with 0.8 Gy irradiation. Spike-in and X-ray respectively, were performed 24hrs before this flow cytometry assessment. Example from TC-1133 3D 194 and irradiation Exp. 10. Figure 4: Flow cytometry assessment of cell composition in ILCs irradiated on day 21 of differentiation, and analyzed one day later. Mono-hormonal beta cells were quantified by NKX6.1 / C-peptide double staining. CHGA positive cells are endocrine cells. Proliferating cells were quantified by Ki67 staining. Cell concentrations determined at the time point of flow cytometry analysis were 2.94xl06cells / ml (control), 2.12xl06cells / ml (0.4 Gy) 2.07xl06cells / ml (0.8 Gy) and 3.29xl06cells / ml (1.2 Gy).

[0079] Figure 5: End-stage ILCs were pre-incubated in low glucose medium, followed by a switch either to low glucose (3 mM) or low glucose plus 25 mM KC1 medium. Non-irradiated ILCs were compared with ILCs that were subjected to X-ray irradiation with 0.8 Gy prior to the secretion assay. Neither basal nor stimulated insulin secretion was affected by irradiation.

[0080] Figure 6: Imaging of end-stage (equivalent of day 23) ILCs generated by thawing and fully differentiating day 16 intermediate stage cryopreserved ILCs until the equivalent of day 23, followed by X-ray irradiation with the indicated doses. ILCs were imaged 2 hours and 24 hours after irradiation. Scale bars are 1000 pm (4x) and 400 pm (lOx).

[0081] Figure 7: Flow cytometry analysis of end-stage clusters (day 23) treated with 0, 0.8 and 10 Gy X-ray irradiation on day 23, and then analyzed 24 hours later after dissociation and staining. Expression of key pancreatic and islet endocrine markers are quantified, as well as SOX9 and Ki67 as progenitor / ductal and proliferation marker, respectively.

[0082] Figure 8: Imaging of end-stage (equivalent of day 23) ILCs after X-ray irradiation with the indicated doses. ILCs were imaged 2 hours, 24 hours and 48 hours after irradiation. Scale bars are 1000 pm (4x) and 400 pm (lOx).

[0083] Figure 9: Cell viability after irradiation of ILCs with different doses and at different time points. Bar plots show the viability % in the order 0 Gy, 0.8 Gy, 4 Gy and 10 Gy for each time point. ILCs were dissociated to single cells before analysis. Data from MO 13 experiment.

[0084] Figure 10: Analysis of double stranded DNA break damage after irradiation using flow cytometry quantification of ILC cells positive for pH2AX. (A) shows data for end-stage ILCs generated from cryopreserved late-stage intermediate material (MO 10 experiment), bars plots show percentage of pH2AX positive cells after treatment with 0 Gy, 0.8 Gy and 10 Gy in that order. (B) shows data from end-stage ILCs generated without an intermittent cryopreservation step (MO 13 experiment). Time points after irradiation and X-ray doses are indicated.

[0085] Figure 11: qPCR of mRNA expression of SASP markers MMP1, CXCL1 and MMP3 in X-ray irradiated end-stage ILCs 24 and 48 hours after irradiation. Bar plots are shown in the order hMMPl, hCXCLl, hMMP3 for each condition. Data from Experiment MO13.

[0086] Figure 12: qPCR of mRNA expression of SASP marker SERPIN El in X-ray irradiated endstage ILCs. Panel A shows data from Experiment MO 10 in ILCs generated from frozen intermediate, at 24 hours post irradiation with the indicated doses. Panel B shows data from Experiment MO 13 at 24 and 48 hours post irradiation with the indicated doses.

[0087] Figure 13: qPCR analysis of inflammatory cytokine mRNA expression in X-ray irradiated (0, 0.8, 4 and 10 Gy) end-stage ILCs. Shown are time points 2 and 24 hours post-irradiation. Expression of CXCL8 is shown in A, Panel B shows expression of IFNB1 and IFNG expression is shown in Panel C. None of the three factors was expressed or modulated to a significant extent at 48 hours (not shown).

[0088] Figure 14: qPCR analysis of proliferation marker and cell cycle regulator expression in cryopreserved intermediate-derived end-stage, X-ray irradiated (0, 0.8 and 10 Gy) ILCs. Data are from Experiment MO10. Panel A shows expression of proliferation markers CDK1, Ki67 and TOP2A. Panel B shows expression of cell cycle inhibitor CDKN1A. Panel B shows expression of non-coding RNA DINO. All data were obtained 24 hours after irradiation.

[0089] Figure 15: qPCR analysis of proliferation marker and cell cycle regulator expression in endstage, X-ray irradiated (0, 0.8, 4 and 10 Gy) ILCs. Data are from Experiment MO 13. Panel A shows expression of proliferation markers CDK1, Ki67 and TOP2A. Panel B shows expression of cell cycle inhibitor CDKN1A. Panel B shows expression of non-coding p53-responsive and -activating RNA DINO. All data were obtained 24 hours after irradiation.

[0090] Figure 16: Schematic representation of a positive feedback loop triggered by low-dose X-ray irradiation, enabling a persistent proliferation block even after initial limited DNA damage caused by low-dose X-ray irradiation has been repaired. Figure 17: Panel A shows random fed blood glucose levels in streptozotocin (STZ)-diabetic immunodeficient NXG mice transplanted with ILC grafts containing 1.5 million monohormonal beta cells (control) or 1.3 million monohormonal beta cells (0.8 Gy group). ILCs in the 0.8 Gy group received X-ray irradiation with the corresponding dose prior to transplantation. Time points of STZ administration, sampling for quantification of circulating human C-peptide (c-pep) and oral glucose tolerance tests (oGTTs) are indicated. Panel B shows circulating human C-peptide levels in random-fed animals from both groups at 2, 6 and 12 weeks after transplantation. Individual values and average values are shown. Data are from EVO22099 study.

[0091] Figure 18: Glucose profiles from oGTTs in the EVO22099 in vivo study, comparing glucose clearance responses in animals with non-irradiated control ILCs vs. responses in animals with transplanted 0.8 Gy X-ray irradiated ILCs after administration of an oral glucose bolus. Data from 4, 8 and 12 weeks after transplantation are shown.

[0092] Figure 19: Shown are kidneys containing ILC grafts explanted from the control group at the end of the Evo22099 in vivo study. Individual animal numbers are shown; grafts appear as lightcoloured areas under the kidney capsule. For animals that had to be dissected prior to the scheduled end of the study at one year due to non-graft related reasons, the day of dissection is shown.

[0093] Figure 20: Shown are kidneys containing ILC grafts explanted from the 0.8 Gy group at the end of the Evo22099 in vivo study. Individual animal numbers are shown; grafts appear as lightcolored areas under the kidney capsule. For animals that had to be dissected prior to the scheduled end of the study at one year due to non-graft related reasons, the day of dissection is shown.

[0094] Figure 21: Shown are representative sections of kidneys containing non-irradiated control ILC grafts explanted at the end (day 365) of the Evo22099 in vivo study, stained for human mitochondria. Individual animal numbers are shown; grafts appear as human mitochondria- and hormone-positive areas under the kidney capsule. Boxed areas are enlarged. Scalebars in the overview images are 1000pm. For animals that had to be sacrificed before the end of the study (due to non-graft-r elated findings), the day of study end is shown. Figure 22: Shown are representative sections of additional kidneys containing non-irradiated control ILC grafts explanted at the end (day 365) of the Evo22099 in vivo study, stained for human mitochondria. Individual animal numbers are shown; grafts appear as human mitochondria- and hormone-positive areas under the kidney capsule. Boxed areas are enlarged. Scalebars in the overview images are 1000pm. For animals that had to be sacrificed before the end of the study (due to non-graft-r elated findings), the day of study end is shown.

[0095] Figure 23: Shown are representative sections of kidneys containing 0.8 Gy X-ray irradiated ILC grafts explanted at the end (day 365) of the Evo22099 in vivo study, stained for human mitochondria. Individual animal numbers are shown; grafts appear as human mitochondria- and hormone-positive areas under the kidney capsule. Boxed areas are enlarged. Scalebars in the overview images are 1000pm. For animals that had to be sacrificed before the end of the study (due to non-graft-r elated findings), the day of study end is shown.

[0096] Figure 24: Example for a CK19-positive ductal cyst observed in a control animal (top images). The boxed section is enlarged in the right image. The table below provides an overview of numbers of ductal cysts observed in both groups, and the number of sections that were analyzed in total for each group.

[0097] Figure 25: After transplantation of end-stage ILCs containing 1 million mono-hormonal beta cells per animal into immunodeficient NXG mice (ILCs irradiated prior to transplantation with 0, 0.4 and 0.8 Gy), circulating human C-peptide levels were determined 2, 6 and 12 weeks after transplantation in random fed animals. Individual values and group averages are shown for each time point. Data from Evo23006 study.

[0098] Figure 26: Shown are kidneys containing ILC grafts explanted from control group (0 Gy) mice at the end of the Evo23006 in vivo study after three months. Individual animal numbers are shown; grafts appear as light-coloured areas under the kidney capsule; arrows point at non- endocrine cell growth in grafts.

[0099] Figure 27: Shown are kidneys containing ILC grafts explanted from X-ray group (0.4 Gy) mice at the end of the Evo23006 in vivo study after three months. Individual animal numbers are shown; grafts appear as light-coloured areas under the kidney capsule; arrows point at non- endocrine cell growth in grafts.

[0100] Figure 28: Shown are kidneys containing ILC grafts explanted from X-ray group (0.8 Gy) mice at the end of the Evo23006 in vivo study after three months. Individual animal numbers are shown; grafts appear as light-coloured areas under the kidney capsule; arrows point at non- endocrine cell growth in grafts.

[0101] Figure 29: Histological analysis of EV023006 control animal #1. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KI: kidney; GR: graft; asterisk: examples for ductal cysts; m: mesenchymal region. The border between kidney and graft is highlighted by dashed lines.

[0102] Figure 30: Histological analysis of Evo23006 control animal #2. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KI: kidney; GR: graft; asterisk: examples for ductal cysts; m: PDX1- negative, putative mesenchymal region. The border between kidney and graft is highlighted by dashed lines. Examples for proliferating cells in ductal cyst epithelium are highlighted with arrows. Examples for proliferating cells in the PDX1 -negative, putative mesenchymal region are highlighted by thin arrows.

[0103] Figure 31: Histological analysis of Evo23006 control animal #3. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KI: kidney; GR: graft; asterisk: examples for ductal cysts; m: PDX1- negative, putative mesenchymal region. The border between kidney and graft is highlighted by dashed lines. Examples for proliferating cells in ductal cyst epithelium are highlighted with arrows. Figure 32: Histological analysis of Evo23006 control animal #4. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KE kidney; GR: graft; asterisk: examples for ductal cysts; m: PDX1- negative, putative mesenchymal region. The border between kidney and graft is highlighted by dashed lines. Examples for proliferating cells in ductal cyst epithelium are highlighted with arrows.

[0104] Figure 33: Histological analysis of Evo23006 control animal #5. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KE kidney; GR: graft; the border between kidney and graft is highlighted by dashed lines.

[0105] Figure 34: Histological analysis of Evo23006 control animal #6. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KE kidney; GR: graft; asterisk: examples for ductal cysts; m: PDX1- negative, putative mesenchymal region. The border between kidney and graft is highlighted by dashed lines. Examples for proliferating cells in ductal cyst epithelium are highlighted with arrows.

[0106] Figure 35: Histological analysis of Evo230060.8 Gy animal #13. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KE kidney; GR: graft; asterisk: example for ductal cysts. The border between kidney and graft is highlighted by dashed lines.

[0107] In the single ductal cyst observed, none of the epithelial cells lining the cyst are KI67-positive. No PDX1 -negative, proliferative areas are apparent. Figure 36: Histological analysis of Evo230060.8 Gy animal #14. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. Putative mesenchymal cells are human mitochondria + / PDX1- / NKX6.1-. KI: kidney; GR: graft; the border between kidney and graft is highlighted by dashed lines. No ductal cysts or proliferative PDX1 -negative areas are apparent.

[0108] Figure 37: Histological analysis of Evo230060.8 Gy animal #15. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. KI: kidney; GR: graft; the border between kidney and graft is highlighted by dashed lines. Only rare, single CK19 positive cells are seen; no ductal cysts or proliferative PDX1 -negative areas are apparent.

[0109] Figure 38: Histological analysis of Evo230060.8 Gy animal #16. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. KI: kidney; GR: graft; the border between kidney and graft is highlighted by dashed lines. Only rare, single CK19 positive cells are seen; no ductal cysts or proliferative PDX1 -negative areas are apparent.

[0110] Figure 39: Histological analysis of Evo230060.8 Gy animal #17. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. KI: kidney; GR: graft; the border between kidney and graft is highlighted by dashed lines. Only rare, single CK19 positive cells are seen; no ductal cysts or proliferative PDX1 -negative areas are apparent. A cavity visible in the enlarged area of the hMito stain does not have KI67-positive cells in its lining, is CK19 negative and lined with bright NKX6.1 -positive cells, demonstrating that it is not a ductal cyst.

[0111] Figure 40: Histological analysis of Evo230060.8 Gy animal #18. HE and human mitochondrial staining provide an overview of organization and extent of the grafted tissue at the end of the experiment. CK19 staining highlights ductal cystic epithelia. NKX6.1 / PDX1 staining highlights pancreatic cells in the graft. KI: kidney; GR: graft; the border between kidney and graft is highlighted by dashed lines. No ductal cysts or proliferative PDX1 -negative areas are apparent.

[0112] Figure 41: Ductal cyst and mesenchymal growth detection assay - overview of growth phase. Shown are Matrigel-embedded, ductal -enriched ILCs on 7 consecutive days after embedding and supply with a suitable growth medium. Prior to embedding, ILCs were X-ray irradiated with 0, 0.8, 4 and 10 Gy. Scale bars are 1000 pm; arrows highlight examples of ductal cysts.

[0113] Figure 42: Ductal cyst and mesenchymal growth detection assay - overview after one additional day in the presence of cAMP enhancers to inflate ductal cysts. Scale bars are 1000 pm in 4x images and 400 pm in lOx images. No ductal cysts are observable with the 4 and 10 Gy X-ray doses.

[0114] Figure 43: Ductal cyst and mesenchymal growth detection assay - low-magnification overview of entire wells after one additional day in the presence of cAMP enhancers to inflate ductal cysts. There are duplicate wells for each condition.

[0115] Figure 44: Imaging of ILCs derived from EN-stage / dayl6 intermediate ILCs that were irradiated in the cryopreserved state, and subsequently taken through final differentiation. Clusters are shown immediately after thaw, and on days equivalent to days 16+1, 16+4 and 16+7. Scale bars are 100 pm.

[0116] Figure 45: Cell viability (A) and cell recovery (B) of ILCs derived from EN-stage / dayl6 intermediate ILCs that were irradiated in the cryopreserved state, and subsequently taken through final differentiation. Time points are immediately after thaw, and on days equivalent to days 16+1, 16+4 and 16+7.

[0117] Figure 46: Flow cytometry analysis of ILCs derived from EN-stage / dayl6 intermediate ILCs that were irradiated in the cryopreserved state, and subsequently taken through final differentiation on day 16+7. Mono-hormonal beta cell fraction (C-peptide / NKX6.1 double positive cells), and expression of key markers PDX1, NKX6.1 and ISL1 were quantified.

[0118] DETAILED DESCRIPTION OF THE INVENTION Definitions

[0119] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0120] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0121] The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.

[0122] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0123] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a compound is defined to be obtainable from a specific source, this is also to be understood to disclose a compound which is obtained from this source.

[0124] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

[0125] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0126] The term “less than” when referring to a specific parameter includes the parameter it is used for, unless otherwise indicated. Hence, “less than 4 Gy” means “equal to or less than 4 Gy”. Similarly, ranges of parameters include their endpoints.

[0127] A “pluripotent stem cell” or “PSC” as used herein refers to a stem cell that has the potential to differentiate into any of the three germ layers, i.e. endoderm, mesoderm or ectoderm, but not into extra-embryonic tissues, i.e. placenta or yolk sac. The term “PSC” or “PSCs” as used herein generally refers to a plurality of cells unless it is explicitly stated that a single cell is meant. Markers for undifferentiated PSC include 0CT4 (P0U5F1, POU class 5 homeobox 1), NANOG (Nanog homeobox), LIN28A (lin-28 homolog A), ESRG (embryonic stem cell related), SOX2 (SRY-box transcription factor 2), ALPL (alkaline phosphatase, biomineralization associated), SSEA3 (Stage-specific embryonic antigen 3), SSEA4 (Stagespecific embryonic antigen 4), TRA-1-60 (Tumor rejection antigen 1-60) and TRA-1-81 (Tumor rejection antigen 1-81). TRA-1-60 and TRA-1-81 are monoclonal antibodies that recognize specific carbohydrate epitopes on the surface of PSCs. SSEA3 and SSEA4 are cell surface glycosphingolipids expressed by stem cells at specific stages of embryonic development.

[0128] The term “induced pluripotent stem cell” or “PSC” as used herein refers to a type of PSC generated directly from a somatic cell by introduction of pluripotency-associated genes, so- called reprogramming factors, such as OCT4 (POU5F1, POU class 5 homeobox 1), SOX2 (SRY-box transcription factor 2), KLF4 (Kriippel-like factor 4) and MYC (MYC protooncogene, bHLH transcription factor).

[0129] In one embodiment, the PSC is a mammalian PSC, preferably a human iPSC or human ESC.

[0130] In one embodiment, the PSC is a iPSC cell line selected from the group consisting of iPSC6.2 / GibcoEpi, iPSl l, iPS15, F002.1A.13, hiPSC-1, hiPSC-2, LiPSC-GRl.l, LiPSC- GR1.2, HEL24.3, HELI 13.5 -corrected, CGT-RCiB-10, MHHiOOl-A, MHHi006-A, MHHi008-A, MHHi008-B, MHHi008-C, VC645-9, VC913-5, VC618-3, VC646-1, iPS 1016, iPS 1031, QHJI01 / -14, RWMH09 / -15 / -23, DRXT18 / -28, RJWI, KTRH05 / -26, D2L01, and D2L03.

[0131] Preferably, it is a clinical grade or GMP -grade iPSC line. It should be noted that iPSC lines can also be generated in a patient-specific manner for personalized / autologous applications, preferably as GMP-grade / clinical grade lines.

[0132] Similarly, many hESC lines, including also clinical / GMP -grade hESC lines are available for use in context with the invention, such as Hl, H9, HUES8, HAD-C100 / -102 / -106, CyT49, MELI, KCL031 / -33 / -34 / -37 / -38 / -40, KARO1, KthESl 1 / -12 / -13 / -14 / -15, RC-09 / -11 / -12 / -13 / - 14 / - 15 / - 16 / - 17, 16, MAN10 / -11 / -12, MAN14 / -15 / -16, ESI-013 / -014 / -017 / -051 / -027 / -035 / - 049 / -053, MasterShef2 / -4 / -10 / -l l, Q-CTS-hESC-l / -2, Yazd4 / -5 / -6 / -7. Preferably, it is a clinical grade or GMP -grade ESC line. The term “proliferative off-target cell” as used herein refers to a fraction of cells that can give rise to unwanted proliferative cell masses arising from PSC-derived therapeutic cells, especially PSC-derived pancreatic islet-like cell clusters (ILCs). They can arise from cells that make unwanted cell fate decisions at the various developmental decision points during an in vitro differentiation protocol. For example, proliferative off-target cell can be cells that do not adopt a terminally differentiated cell pancreatic endocrine cell fate during in vitro differentiation, for example because they remain at a multipotent progenitor stage. They can also consist of cells that either have not adapted a pancreatic fate, and therefore commonly do not express the pancreatic markers PDX1 and NKX6.1. They may also arise by trans-differentiation of pancreatic cells to a mesenchymal -like phenotype. The most common proliferative off-target cells in PSC-derived ILCs are mesenchymal cells, pancreatic progenitor cells and ductal cells. Potentially less common but highly impactful if they occur are residual pluripotent stem cells.

[0133] The term “unwanted cell growth” as used herein refers to growth of cell masses originating from proliferative off-target cells. In this context, cells that make unwanted cell fate decisions at the various developmental decision points during an in vitro differentiation protocol means that these cell do not differentiate towards the intended cell fate that is the goal of the in vitro differentiation protocol.

[0134] The term “mesenchymal cell” as used herein refers to proliferative cells that are PDX1 -negative and non-pancreatic. Mesenchymal cells arise through conversion of non-endocrine pancreatic progenitor cells after implantation. Mesenchymal cells are negative for key pancreatic markers such as PDX1, and are also negative for NKX6.1. Mesenchymal cells have the capacity to form mesenchymal cell masses. Mesenchymal cells may also arise already in vitro during differentiation from PSC to ILC stage. Other (non-mesenchymal) PDX1- negative cells may be present and contribute to the growth of cell masses.

[0135] PDX1 (pancreatic and duodenal homeobox 1) is a transcription factor necessary for pancreatic development, including beta cell maturation. PDX1 expression is required for the maintenance and survival of beta cells, and thus can be used as a marker of the pancreatic lineage and mature beta cells.

[0136] NKX6. l is a transcription factor required for the development of pancreatic beta cells. Hence, NKX6.1 can be used as a marker of the pancreatic lineage and mature beta cells. Mono- hormonal beta cells (in contrast to polyhormonal cells, insulin-positive cells also expressing other hormones such as glucagon, which do not give rise to functional beta cells after full maturation) are C-peptide / Insulin and NKX6.1 double positive.

[0137] The term “ductal cell” as used herein refers to CK19-positive cells that are proliferative and form expanding ductal cysts in iPSC-derived ILCs. Proliferating multipotent pancreatic progenitors which are able to give rise to ductal cells are generated in all differentiation protocols for ILCs, since progenitors are an obligatory intermediate stage also on the path towards endocrine cells. Expanding ductal cysts are likely a manifestation of residual early pancreatic progenitors in the graft, and not of terminally differentiated ductal cells. Ductal cysts enlarge over time, and can become very large in longer-term in vivo studies. Pancreatic ductal epithelial cells transport alkaline fluid to the luminal side, and thereby may contribute to volume expansion of cysts. Long-term cyst expansion is expected to be dependent on cyst epithelial proliferation to accommodate the required enlargement of epithelial area lining the cyst.

[0138] CK19 (cytokeratin 19, hKRT19) is a marker of epithelial cell differentiation and is specific for undifferentiated pancreatic ductal cells as well as mature ductal cells. CK19 is especially useful to detect ductal cysts in histological sections of ILC grafts, for example after retrieval from animals in in vivo validation studies.

[0139] Ki-67 is a marker of proliferation that is present during all active phases of the cell cycle.

[0140] The term “residual” when referring to pluripotent stem cells (PSCs) as used herein refers to PSCs that are undifferentiated and remain pluripotent, i.e. retain the capacity to proliferate and differentiate into different cell types, within a population of differentiated cells. The population of differentiated cells may be e.g. an islet-like cluster. The islet-like cluster may form part of a transplant for use in treatment. Hence, a transplant may comprise PSC-derived islet-like clusters and residual PSCs. The residual PSCs have the capacity to differentiate into different cell types and to form cell masses, such as teratomas.

[0141] The term “islet-like cluster” as used herein, also known in the literature as “islet-like organoids” or “islet-like cell cluster” refers to aggregates of pancreatic [3- and other pancreatic endocrine cells derived from pluripotent stem cells. Pancreatic islets or islets of Langerhans are regions of the pancreas that contain endocrine cells. In order to recreate the structure and function of pancreatic islets (“islet-like clusters”), pluripotent cells are differentiated step-wise and in a directed manner in vitro, first into definitive endoderm (DE, expressing e.g. SOX17 and CXCR4 / EPCAM), followed by differentiation into primitive foregut (PF, expressing e.g. F0XA2), pancreatic endoderm and pancreatic progenitors (PPI and PP2 stage, expressing PDX1 and PDX1 / NKX6.1, respectively) and finally differentiated into aggregates of endocrine pancreas cells including pancreatic P-cells by modulating notch signalling through gamma secretase inhibition. Endocrine cells are characterized by expression of Chromogranin A and other pan-endocrine markers, and also express markers characteristic for the specific cell types. A detailed description of a protocol used for directed differentiation of PSCs into ILCs can be found in W02023203208.

[0142] Mono-hormonal beta cells are C-peptide / NKX6.1 double positive. Acquisition of double positive status is equivalent to a stable (3-cell fate decision. Mono-hormonal beta cells mature, in vitro and / or in vivo / m' patients through a fetal-like immature stage, in which cells secrete insulin in response to membrane depolarization (e.g. with KC1) but not in response to elevated (>5.6 mM) levels of glucose, to a mature stage in which cells become responsive to elevated glucose levels. Maturation can proceed further so that cells acquire expression of additional adult beta cells markers such as SIX2 and SIX3, and also achieve an overall pharmacological and electrophysiological response profile as found in P-cells in adult human donor islets.

[0143] “End-stage ILCs” is defined as a state in which most or all cells in in vztro-differentiated cell population have completed their endocrine cell fate decision, and where only small changes in the fraction of monohormonal beta cells are observed without additional intervention.

[0144] “EN-stage” or “EN-stage ILCs”, “late intermediate stage ILCs” or “day 16” ILCs are ILCs that are at or near the middle of the endocrine differentiation process triggered by gamma secretase addition. In the context of the differentiation process used in the current application, this corresponds to day 16 after initiation of differentiation.

[0145] The terms “transplant”, “implant” and “graft” are used herein interchangeably and refer to the a PSC-derived ILC before or after being transferred into a subject, i.e. while being cultured in vitro in preparation for being implanted into the subject, or afterwards when in situ, grafted onto the tissue of the subject. The term “low-dose X-ray” as used herein refers to an absorbed dose of electromagnetic radiation. Ionizing radiation deposits energy when penetrating objects or tissues, the energy absorbed from exposure to radiation is referred to as a “dose”. The absorbed dose, e.g. absorbed by cells, tissues and / or the human body, is measured with the SI unit gray (Gy). One gray dose is equivalent to one joule of radiation energy absorbed per kilogram of tissue weight. Rad is the CGS unit for absorbed radiation and 1 rad = 0.01 Gy = O.OlJ / kg. As used herein, low-dose X- ray refers to doses of less than 10 Gy, preferably equal or less than 4 Gy.

[0146] The term “cell therapy” as used herein refers to the transplantation, e.g. injection, of human cells to replace or repair damaged tissue and / or cells, also known as “cellular therapy” or “cell transplantation”. Cell therapy can be allogeneic (allotransplantation), which means that the cell therapy donor is a different subject as the recipient. The term “allogenic” is also applied if the therapeutic cells are generated from a PSC line that has not been generated by reprogramming somatic cells of the recipient into iPSC. Autologous cell therapy (autotransplantation) refers to cases where the transplanted cells are derived from the patient’s own tissue. The term “autologous” can also be applied if a somatic cell of a patient is reprogrammed into an iPSC, followed by directed differentiation into a therapeutic cell type(s). Xenogeneic cell therapy (xenotransplantation) refers to cell therapies wherein the patient receives cells from another species, e.g. a human receiving pig cells. In “PSC-based” cell therapy, therapeutic cells are generated from pluripotent stem cells. In one embodiment, the cell therapy of the invention may be autologous or allogeneic PSC-based cell therapy.

[0147] The term “improved safety profile” when referring to PSC-derived ILCs herein means that the likelihood of unwanted or uncontrolled cell growth is reduced compared of untreated PSC- derived ILCs, thereby reducing the risk of complications for the patient.

[0148] The terms “reducing”, “eliminating” and “arresting”, as used herein, refer to the result of the low-dose X-ray irradiation treatment, which eliminates any significant proliferative off-target proliferative cell mass formation (as detectable in suitable in vitro assays and after transplantation) of PSC-derived ILCs. Even in long term experiments, no residual proliferative off-target cells or cell masses could be detected in vivo after low-dose -X-ray irradiation. The terms reducing and arresting are used in addition since the absence of proliferating mass formation after irradiation can also, or in addition, be due to either reducing mass-forming cells below a critical threshold. The term “prior to treatment” as used herein when referring to the step or irradiation of ILCs means that the irradiation is carried out ex vivo before ILCs are transplanted into a subject. The irradiation is not carried out in vivo, such as after transplantation into the subject.

[0149] The term “long-term DNA damage” as used herein refers to DNA damage, such as doublestrand breaks, that is not transient, i.e. that is not repaired. Accumulated DNA damage can give rise to genomic instability and induce signaling cascades leading to cell senescence, induction of innate inflammatory cytokine expression, or cell death. Long-term DNA damage such as unrepaired DNA double-stranded breaks can be detected by phospho-H2AX / yH2AX (phosphohistone H2A.X) staining.

[0150] The term “senescence” as used herein refers to a stress response elicited by DNA damage that is characterized by cell cycle arrest and an immunogenic phenotype, also called senescence- associated secretory phenotype. Levels of the protein PALI, encoded by SERPINE1, can be used as a marker of senescence, since PALI can be a component of the senescence-associated secretory phenotype (SASP) and can induce cellular senescence. Other SASP markers include MMP1 (matrix metalloproteinase 1), CXCL1 (chemokine (C-X-C motif) ligand 1), and MMP3 (matrix metalloproteinase 1).

[0151] The terms “innate cytokine expression”, “innate inflammatory cytokine expression” or “innate inflammatory marker expression” refer to the radiation-induced induction of certain cytokines and gene expression profiles that has been reported as induced after irradiation of cultured cells with X-ray doses e.g. of 20 Gy (Tigano et al., Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance. Nature. 2021 Mar;591(7850):477-481. PMID: 33627873.). Key markers and inflammatory cytokines are in this context IFNB1, IFNG, the interferon signaling marker gene ISG15, and CXCL8. Secretion of interferons and / or CXCL8 and related factors can lead to the attraction and activation of immune cells and the creation of an inflammatory environment in and around a cell therapy graft, and can thereby reduce or prevent the function and survival of grafted cells (Sintov et al., Whole-genome CRISPR screening identifies genetic manipulations to reduce immune rejection of stem cell-derived islets. Stem Cell Reports. 2022 Sep 13;17(9):1976-1990. PMID: 36055241; PMCID: PMC9481918.). CXCL8 / IL8 is an inflammatory cytokine known to attract and / or activation of neutrophils and myeloid cells, with adverse consequences for graft survival and function (Ehses et al., Increased number of islet- associated macrophages in type 2 diabetes. Diabetes. 2007 Sep;56(9):2356-70. PMID: 17579207.; Citro et al., CXCR1 / 2 inhibition enhances pancreatic islet survival after transplantation. J Clin Invest. 2012 Oct; 122(10): 3647-51. PMID: 22996693; PMCID: PMC3461913.; Naziruddin et al., Evidence for instant blood-mediated inflammatory reaction in clinical autologous islet transplantation. Am J Transplant. 2014 Feb;14(2):428-37. PMID: 24447621.).

[0152] The terms “ILC cell maturation” and “ILC cell function” as used herein refer to beta cell maturation and function in the ILC transplant. At the time of transplantation, ILCs typically contain immature beta cells, which mature to acquire the functions of full glucose responsiveness and insulin content after implantation. One marker of maturation of beta cells is the level of human C-peptide. However, conditions and timing of ILC maturation can be adjusted to generate ILCs with fully matured P-cells, indicated e.g. by a substantial insulin secretory response to elevated glucose levels.

[0153] The term “composition” as used herein refers to a mixture comprising therapeutically effective PSC-derived ILC cells according to the present invention, i.e. an ILC preparation that has been subjected to low-dose X-ray irradiation, and which is suspended in a suitable medium and buffer containing additional supplements or additives to provide a survival and function preserving and promoting environment prior to administration. Media, excipients, supplements and additives need to be non-toxic, safe and compliant with regulatory requirements for cell therapeutic agents. Transport and administration can either occur with low-dose X-ray irradiated ILCs being suspended in the same medium, or a wash / replacement step with transfer into a dedicated administration medium can be integrated into the workflow, as also discussed in Weng L. Cell Therapy Drug Product Development: Technical Considerations and Challenges. J Pharm Sci. 2023 Oct;112(10):2615-2620. PMID: 37549846; AtoufF. Cell-Based Therapies Formulations: Unintended components. AAPS J. 2016 Jul;18(4):844-8. PMID: 27233803; Zhang et al., Formulation strategies in immunotherapeutic pharmaceutical products. World J Clin Oncol. 2020 May 24; 11 (5):275-282. PMID: 32728530.

[0154] Methods of the invention In a first aspect, the invention provides a method of reducing and / or eliminating unwanted cell growth in PSC-derived islet-like clusters (ILCs) by low-dose X-ray irradiation in vitro, comprising a) obtaining a population of cells comprising PSC-derived ILCs and proliferative off-target cells; and b) irradiating the population of cells with low-dose X-ray, wherein the low-dose is less than 10 Gy.

[0155] ILCs can be generated by differentiating PSCs according to the “Second Protocol” described in WO 2023 / 203208. Suitable ILCs can also be generated using other published protocols for the directed differentiation of PSCs into ILCs in suspension culture. Differentiation can also be performed with parts of the procedure being performed in 2D culture followed by dissociation and re-aggregation into 3D aggregates.

[0156] ILCs generated with the Second Protocol can, for example, be irradiated with low-dose X-rays on day 16 of the differentiation procedure (EN stage), and then differentiated further until harvest and transplantation on day 23.

[0157] Alternatively, ILCs can be frozen on day 16 (EN stage) using methods described in WO 2023 / 203208, and frozen ILCs can be irradiated with low-dose X-rays. Irradiated clusters can either be thawed directly to complete differentiation, or returned to cryo-storage to be thawed at a later time. After thaw, the irradiated EN-stage ILCs are differentiated further, typically but not limited to until the equivalent of day 23, when they can be harvested for transplantation.

[0158] The timepoint of harvest after EN-stage irradiation and thaw, or after irradiation of non-frozen EN-stage ILCs can be chosen flexibly, with harvest and transplantation e.g. also on the equivalent of day 20, day 21, day 22, day 24, day 25, day 26, or later time points, e.g. depending on the desired degree of cell maturation. Clusters comparable to the day 16 EN-stage clusters described above may arise at deviating time points in the context of other differentiation protocols, for example on day 14 or on day 17, but may be comparable in that they are at a comparable stage with substantial but incomplete endocrine differentiation. EN-stage ILCs may also be dissociated before cryopreservation, and reaggregated after thaw. Low-dose X-ray irradiation can also be applied to cryopreserved single cell suspensions of ILC cells.

[0159] ILCs may also be frozen at EN stage, thawed and taken through final differentiation, e.g. as described in WO 2023 / 203208, harvested, irradiated with low-dose X-rays, before transplantation. Harvesting can be at the equivalent of day 23 of differentiation, or in at time frame of 20 to 28 days or beyond.

[0160] ILCs may also be treated with low-dose X-ray irradiation without prior cryopreservation, or after clusters were cryopreserved with another method, e.g. by irradiating ILC cells frozen as a single cell suspension and re-aggregation post thaw, or by irradiating live ILCs generated by freezing single cell suspension at / around EN-stage, post-thaw reaggregation and differentiation, and irradiation at or near end-stage.

[0161] In a second aspect, the invention provides an in vitro method of producing PSC-derived ILCs with improved safety profile suitable for cell therapy, the method comprising a) producing ILCs by culturing PSC under conditions suitable for the formation of ILCs; and b) irradiating the ILCs produced in step a) with low-dose X-ray, wherein the low-dose is less than 10 Gy.

[0162] In one embodiment, the method further comprises a step of culturing the population of PSC- derived ILCs after low-dose X-ray irradiation for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days.

[0163] In one embodiment, the method further comprises a step of monitoring whether the low-dose X-ray irradiated ILCs are free of proliferative off-target cells.

[0164] In one embodiment, monitoring whether the irradiated ILCs are free of proliferative off-target cells comprises determining the expression of one or more proliferation markers or one or more pluripotency markers, preferably wherein cells positive for one or more proliferation marker are reduced compared to controls that were not irradiated.

[0165] In one embodiment, the one or more proliferation or pluripotency markers are selected from CDK1 or other cell-cycle related genes or cell-cycle related protein modifications, PCNA, Ki67, T0P2A (DNA toposisom erase II alpha), incorporation of EdU, BrdU or similar compounds, ALPL, TRA-1-60, TRA1-80, OCT4, NANOG, SSEA-4 or SOX2.

[0166] In one embodiment, the one or more proliferation or pluripotency markers are selected from the group consisting of CDK1, PCNA, Ki67, TOP2A (DNA toposisom erase II alpha), , ALPL, TRA-1-60, TRA1-80, OCT4, NANOG, SSEA-4, SOX2, and incorporation of EdU or BrdU.

[0167] In one embodiment, the one or more proliferation markers are selected from the group consisting of PCNA, Ki67, CDK1, BrdU incorporation, EdU incorporation, and TOP2A.

[0168] In one embodiment, marker expression is monitored using antibody staining, preferably followed by flow cytometry or immunohistochemistry.

[0169] In another embodiment, monitoring whether the irradiated ILCs are free of proliferative off- target cells comprises detection of proliferative mass forming cells using cell-based assays in vitro. In one embodiment, detection of proliferative cells is performed using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, ductal cyst formation and mesenchymal cell detection assay, or transplantation into immunodeficient animals. Proliferative mass forming cells can also be detected using single cell or single nucleus sequencing, or by detecting unwanted-cell specific transcripts using qPCR, ddPCR or other sensitive methods to detect rare transcripts.

[0170] Monitoring can be performed 2 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours or more after irradiation.

[0171] In another embodiment, monitoring whether the irradiated ILCs are free of proliferative off- target cells comprises detection of proliferative mass forming cells using suitable in vivo models. For example, ILC and other grafts that are tested for the occurrence of unwanted cell growth can be implanted under the kidney capsule of immunodeficient mice, or of immunodeficient rats. Other implantation sites have also been described, e.g. subcutaneously, into fat pads, intramuscularly, or infusion into the liver through the portal vein. To facilitate the analysis of maturation and function of ILCs, recipient animals can be made diabetic prior or after transplantation, e.g. by injection of streptozotocin. However, maturation and function (e.g. via quantification of circulating human C-peptide levels) can also be performed in non-diabetic recipient animals. Besides rodents, ILC preparations, their maturation and function, and whether they give rise to unwanted cell growth or not can also be tested in immunosuppressed pigs, dogs, or non-human primates. Whether or not unwanted cell growth occurs can be determined by non-invasive imaging, palpation, and / or resection / dissection followed by macroscopic evaluation of the explant and histology.

[0172] Off-target proliferative cells and unwanted growth

[0173] Before implanting PSC-derived ILCs into patients, ILCs can be tested in preclinical model systems, typically in immunodeficient mice (to prevent xeno-r ejection), which can be made diabetic by chemically ablating beta cells using the beta cell toxin streptozotocin (STZ). Such models are considered predictive for clinical trials in humans, since it has been shown that isolated human islets can normalize diabetes in this model in a similar manner as in patients; in addition, a comprehensive set of clinically relevant parameters can be assessed (e.g. blood human C-peptide levels, random fed and fasted glucose levels, as well as responses to glucose tolerance tests). Such studies are typically performed as part of ILC manufacturing process optimization activities. Unwanted cell growth sometimes can occur despite an overall high quality (high beta cell and endocrine fraction) flow cytometry profile of the implanted cells. A similar outcome is expected after transplanting corresponding cell batches into patients.

[0174] At least three kinds of unwanted cell growth / cell types are of note: a) ductal cysts and exocrine precursors; b) mesenchymal cells and c) residual pluripotent cells derived from the pluripotent starting material.

[0175] Ductal cysts and proliferating multipotent progenitors presumably arise from pancreatic progenitor cells present in the graft material that had, at the time of implantation, not yet committed to an endocrine fate. Ductal cells in grafts may also derive from cells acquiring a ductal fate already during differentiation. However, based on abundant clinical evidence from human islet transplantation, it can be expected that terminally differentiated ductal cells are not causing problems in patients receiving islet transplants, since transplanted human islet material can contain 50% or more non-islet cells, most of which are ductal epithelial cells (Benomar et al. 2018). There are also no reports of expanding ductal cysts or other unwanted proliferating cell masses (PDX1 negative; mesenchymal) when isolated human islet preparations are transplanted under the kidney capsule. Therefore, expanding ductal cysts are likely a manifestation of residual early pancreatic cells in the graft, and not of terminally differentiated ductal cells. While ductal cysts are not tumors in a strict sense, animal study data show that they can enlarge over time, and can become very large in longer-term in vivo studies (and presumably also in patients). This cannot be tolerated in diabetes patients, who are anticipated to live with ILC implants for many years or decades. Pancreatic ductal epithelial cells transport alkaline fluid to the luminal side, and thereby may contribute to volume expansion of cysts. Long-term cyst expansion is expected to be dependent on cyst epithelial proliferation to accommodate the required enlargement of epithelial area lining the cyst. Therefore, blocking or arresting proliferation of ductal cells or their precursors is a potential strategy to prevent the formation of large cysts. Complete killing and / or elimination of ductal cells and especially proliferating pancreatic progenitors is expected to deliver the same result, but is likely not required. Multipotent pancreatic progenitors (expressing SOX9 and PDX1, Jennings et al. 2015), which are able to give rise to ductal and acinar cells (and possibly committed ductal cells themselves) are generated in all differentiation protocols for ILCs, since such progenitors are an obligatory intermediate stage also on the path towards endocrine cells.

[0176] Graft-derived (= positive for a human cell marker) mesenchymal, non-pancreatic (not expressing PDX1 and other pancreatic markers) cell masses have been observed in some grafts by the inventors and others (Augsornworawat et al. 2020; Chandra et al. 2022; Hiyoshi et al. 2024). These masses in some cases made up 50% or more of the entire graft mass, and contained proliferating areas. Expanding mesenchymal masses are not acceptable in patients since their slow expansion poses a safety risk on several levels.

[0177] Small amounts of mesenchymal cells were already seen by Otonkoski and colleagues when analyzing PSC-derived intermediate stages of ILC differentiation in vitro, with a differentiation trajectory coming from pancreatic precursors (Chandra et al., 2022). Furthermore, Hiyoshi et al. performed an analysis of differentiation trajectories in their single cell data, and also came to the conclusion that mesenchymal cells (called PMSCs in this publication) arise through conversion of non-endocrine pancreatic (PDX1 positive) progenitor cells after implantation.

[0178] By analyzing published single cell sequencing data, Hiyoshi et al. show that the mesenchymal cells / PMSCs observed by them and mesenchymal cells observed by Augsornworawat et al. have a strongly overlapping gene expression profile, and therefore represent the same cell type. Based on marker expression data, these cells also clearly appear to be the same as those seen in the grafts described herein explanted after long-term studies, supporting the conclusion from Hiyoshi et al. that the mesenchymal cells / PMSCs are a universal phenomenon that is observed independently from a particular differentiation process and / or PSC line. Mesenchymal cells are negative for key pancreatic markers such as PDX1, and are also negative for NKX6.1. The presence of proliferating non-pancreatic (=PDX1 -negative) cells in an ILC graft has to be minimized or avoided, regardless of the identity or cell type.

[0179] As pointed out above, due to the nature of the stepwise differentiation process simulating embryonic development, which must pass through a pancreatic progenitor stage on the way to endocrine / beta cells, residual non-endocrine cells are an almost unavoidable component of ILCs generated from PSCs, and therefore the occurrence of mesenchymal outgrowth appears to be a universally relevant problem in PSC-based diabetes cell therapy.

[0180] Residual pluripotent cells remain a difficult-to-detect concern. Already small numbers of cell clusters containing pockets of pluripotent cells may cause tumor formation over time.

[0181] Hence, in one embodiment, the unwanted cell growth arises from proliferative off-target cells selected from the group consisting of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells.

[0182] As stated above, unwanted proliferative off-target cells can lead to ductal cell masses (ductal cysts) expanding mesenchymal masses, and teratomas, posing a safety risk to patients receiving PSC-derived ILC transplants. The method of the invention uses low-dose X-ray irradiation, eliminating and / or greatly reducing proliferative off-target cells in PSC-derived ILCs before transplantation, thereby improving the clinical safety profile of the ILC transplant. The term cell therapy in this regard refers to the transplantation of PSC-derived ILCs into patients.

[0183] The inventors were surprisingly able to show that X-ray doses of less than 10 Gy were sufficient to eliminate unwanted cell growth arising from proliferative off-target cells, without negatively impacting senescence status, inflammatory gene expression, and maturation as well as longterm function of PSC-derived ILC transplants.

[0184] In one embodiment, the ILCs comprise a decreased percentage of i) proliferating multipotent pancreatic progenitors and cells derived thereof being able to form ductal cysts; ii) PDX1 -negative cells, such as mesenchymal cells, and / or iii) residual pluripotent cells; after low-dose X-ray irradiation compared to controls that were not irradiated, wherein the low- dose is between 0.8 Gy and 10 Gy, preferably between 0.8 Gy and 4 Gy.

[0185] In one embodiment, the decreased percentage is at least 10%. In a preferred embodiment, the decreased percentage is at least 50%.

[0186] Hence, in one embodiment, the ILCs comprise at least 10% less of i) proliferating multipotent pancreatic progenitors and cells derived thereof being able to form ductal cysts; ii) PDX1 -negative cells, such as mesenchymal cells, and / or iii) residual pluripotent cells; after low-dose X-ray irradiation compared to controls that were not irradiated, wherein the low- dose is between 0.8 Gy and 10 Gy, preferably between 0.8 Gy and 4 Gy.

[0187] In one embodiment, after low-dose X-ray irradiation the percentage of residual mitotic cells in the PSC-derived ILCs is less than 10%, preferably less than 1%.

[0188] In one embodiment, after low-dose X-ray irradiation, the low-dose X-ray irradiated ILCs of the invention form detectable ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses at lower rates than controls that were not irradiated.

[0189] In one embodiment, after low-dose X-ray irradiation, the low-dose X-ray irradiated ILCs of the invention do not form detectable ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses at least 1 year after transplantation, preferably at least 5 years after transplantation.

[0190] In one embodiment, after low-dose X-ray irradiation, the ILCs of the invention do not form detectable ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, ductal cyst formation and mesenchymal cell detection assay, or transplantation into immunodeficient animals. In one embodiment, the ILCs do not form detectable ductal cysts, mesenchymal cell masses and / or pluripotent cell masses for at least 1 year after implantation, preferably for at least 5 years after implantation, wherein progenitor derived, non-endocrine cell masses, ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, ductal cyst formation and mesenchymal cell detection assay, or transplantation into immunodeficient animals.

[0191] Low-dose X-ray irradiation

[0192] Doses less than 10 Gy, 4 Gy or less, inhibited unwanted cell growth, e.g. formation of ductal cysts and PDX1 -negative cell masses in ILCs, even with very long (up to one year) in vivo observation of grafts, and without negatively impacting maturation or function. Less than 1 Gy (0.8 Gy) was sufficient to efficiently kill or arrest pluripotent cells in vitro.

[0193] In one embodiment, the low-dose X-ray is less than 8 Gy, less than 6 Gy, less than 4 Gy, less than 2 Gy, less than 1 Gy. In one embodiment, the low-dose X-ray is about 0.8 Gy.

[0194] In a preferred embodiment, the low-dose X-ray is less than 4 Gy. In an even more preferred embodiment, the low-dose X-ray is less than 2 Gy.

[0195] In one embodiment, the low does is less than 4 Gy. In one embodiment, the low does is less than 2 Gy. In one embodiment, the low-dose is less than 2 Gy. In one embodiment, the low- dose is less than 1 Gy. In one embodiment, the low-dose is about 0.8 Gy.

[0196] In another embodiment, the low-dose is between 0.8 to 10 Gy. In another embodiment, the low- dose is between 0.8 to 8 Gy. In another embodiment, the low-dose is between 0.8 to 6 Gy. In a preferred embodiment, the low-dose is between 0.8 to 4 Gy. In another embodiment, the low- dose is between 0.8 to 2 Gy.

[0197] Doses given as “less than” or ranges herein include endpoints.

[0198] The applied X-ray dose can be determined, if available, using a built-in dosimetry unit (Multirad 225), and / or with miniature dosimeters that can be placed under the same conditions and in the same location where cells are located. This can be done for example using miniature chip format dosimeters like myOSL chips (Radpro International GmbH, Remscheid, Germany) or with rodshaped TL-detectors (Materialprufungsamt Nordrhein-Westfalen, Dortmund, Germany).

[0199] The above mentioned dose selectively prevented growth of ductal cysts in long-term in vivo studies when ILCs were irradiated either during endocrine commitment or shortly before implantation, while leaving in vivo beta cell maturation and function unchanged.

[0200] Irradiation of cell clusters at endocrine commitment means that cells are irradiated at a stage when they can be harvested and then frozen with high efficiency (see also W02023203208A1). Given the common origin of ductal cysts and mesenchymal cell masses, it can be concluded that the method also has the ability to abrogate the formation of masses of PDX1 -negative (e.g. mesenchymal) cells.

[0201] The present inventors are the first to demonstrate long-term in vivo function of implanted ILCs without ductal cyst or mesenchymal mass formation in an experiment lasting substantially longer than half a year (as shown in the Hiyoshi et al. study).

[0202] The applied dose did not induce long-term DNA damage, as indicated by gamma H2AX staining in flow cytometry.

[0203] The applied dose did not induce expression of the S ASP / senescence marker SERPINE1 / PAI-I (see also d'Adda di Fagagna et al. 2008), indicating that unlike competitor doses, the method of the invention achieves the desired effect without generating a potentially detrimental fibrosis inducing SASP component (Nguyen et al. 2018).

[0204] The applied doses also did not induce significant transient expression of CXCL8 / IL8, meaning that an inflammatory, neutrophil / myeloid cell attracting phenotype was not induced.

[0205] Low dose irradiation also did not induce expression of other innate proinflammatory cytokines or markers such as IFNG, ISG15 or IFNB1, as shown in the literature for high irradiation doses such as 20 Gy (Tigano M, Vargas DC, Tremblay-Belzile S, Fu Y, Sfeir A. Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance. Nature. 2021 Mar;591(7850):477-481. PMID: 33627873.).

[0206] Presently, the inventors have found a method to (selectively) eliminate unwanted in vivo ductal cyst formation in ILC preparations for cell therapy with low-dose X-ray irradiation, without inducing long-term DNA damage, substantial PAI-I and SASP induction, CXCL8 induction, functional or maturation impairment, adverse effects on cell composition (such as a reduction in beta cell fraction) and without using radioactive radiation sources. They were not only able to show that although some DNA damage is induced at the doses used by us, this damage is only transient, but also that the radiation-induced proliferation block persists even after the radiation-induced DNA damage has been repaired. The stages of irradiation tested allow integration of irradiation into ILC manufacturing without additional cell / cluster handling and additional process steps.

[0207] Furthermore, the inventors have adapted a method showing ductal cyst formation in vitro, and have shown irradiation effects in that system.

[0208] Hence, in one embodiment, the invention relates to a method of reducing and / or eliminating proliferative off-target cells in PSC-derived islet-like clusters (ILCs) by low-dose X-ray irradiation in vitro, comprising a) obtaining a population of cells comprising PSC-derived ILCs; and b) irradiating the population of cells with low-dose X-ray, wherein the low-dose is less than 10 Gy; wherein the unwanted cell growth arises from proliferative off-target cells selected from the group consisting of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells.

[0209] In one embodiment, the low-dose X-ray irradiation does not induce

[0210] (i) long-term DNA damage;

[0211] (ii) expression of the senescence markers;

[0212] (iii) expression of inflammatory cytokines, and / or

[0213] (iv) impairment of ILC cell composition, cell maturation or function.

[0214] In one embodiment, long-term DNA damage is measured by determining the expression of pH2AX. The term long-term DNA damage refers to DNA damage that is not transient. In one embodiment, low-dose X-ray irradiation does not induce one or more senescence markers selected from the group consisting of SERPINE1 / PAI-I, MMP1, CXCL1 and MMP3.

[0215] In one embodiment, low-dose X-ray irradiation with a dose between 0.8 - 4 Gy does not induce

[0216] (i) long-term DNA damage;

[0217] (ii) expression of the senescence markers, preferably selected from the group consisting of SERPINE1 / PAI-I, MMP1, CXCL1 and MMP3;

[0218] (iii) substantial expression of CXCL8 / IL8 and / or

[0219] (iv) impairment of ILC cell composition, cell maturation or function.

[0220] In one embodiment, ILC cell maturation is determined by determining the levels of one or more hormone markers selected from the group consisting of C-peptide, glucagon (GCG), somatostatin (SST) and serotonin (5-HT) and / or one or more pancreatic cell markers selected from the group consisting of PDX1 (pancreatic and duodenal homeobox 1), ISL1 and NKX6.1 (NK6 homeobox 1). In one embodiment, pancreatic cells are PDXl-positive and NKX6.1- positive.

[0221] In one embodiment, ILCs consist mainly of pancreatic endocrine cells, characterized by the expression of chromogranin A (CHGA), and the expression of other markers typical for pancreatic endocrine cells, such as synaptophysin.

[0222] In one embodiment, a substantial fraction of endocrine cells in ILCs are mono-hormonal beta cells, defined as cells co-expressing C-peptide / insulin and NKX6.1.

[0223] In one embodiment, ILC quality and cell function is determined by measuring KCl-induced insulin secretion in vitro., and / or by measuring overall cell yield; and / or by measuring percentage of beta-cells; compared to controls that were not irradiated. Analysis of cell composition can be done by flow cytometry using fluorescent antibody staining and detection. Testing of beta cell function can also be done by assessing glucose stimulated insulin secretion, response to various secretagogues (such as sulfonylureas and ion channel modulators) and by assessing insulin processing. In one embodiment, low-dose X-ray irradiation supresses the unwanted growth of ductal cysts, mesenchymal cell masses and / or residual pluripotent cell masses in the ILCs. Preferably, the unwanted growth occurs after transplantation of the ILCs.

[0224] In one embodiment, low-dose X-ray irradiation with about 0.8 Gy completely eliminates residual pluripotent PSCs in PSC-derived ILCs.

[0225] In one embodiment, low-dose X-ray irradiation with about 0.8 Gy completely eliminates residual pluripotent PSCs in non-frozen PSC-derived ILCs.

[0226] In one embodiment, low-dose X-ray irradiation with less than 10 Gy completely eliminates residual pluripotent PSCs in PSC-derived ILCs.

[0227] In one embodiment, low-dose X-ray irradiation with less than 10 Gy completely eliminates residual pluripotent PSCs in non-frozen PSC-derived ILCs.

[0228] In one embodiment, low-dose X-ray irradiation with equal or less than 4 Gy completely eliminates residual pluripotent PSCs in PSC-derived ILCs.

[0229] In one embodiment, low-dose X-ray irradiation with equal or less than 4 Gy completely eliminates residual pluripotent PSCs in non-frozen PSC-derived ILCs.

[0230] In one embodiment, low-dose X-ray irradiation with about 0.8 Gy i) eliminates ductal cyst formation; ii) eliminates pluripotent cells in vitro, preferably by inducing growth arrest; iii) does not cause long-term damage to DNA; iv) does not induce a SASP phenotype in ILCs, such as expression of PAI-l / SERPIN El; v) does not induce substantial transient expression of CXCL8 / IL8, vi) suppresses the expression of proliferation markers, such as CDK1, KI67 and TOP2A; and / or vii) does not negatively affect beta cell fraction, maturation and / or function.

[0231] In one embodiment, low-dose X-ray irradiation with less than 10 Gy i) eliminates ductal cyst formation; ii) eliminates pluripotent cells in vitro, preferably by inducing growth arrest; iii) does not cause long-term damage to DNA; iv) does not induce a SASP phenotype in ILCs, such as expression of PAI-l / SERPIN El; v) does not induce substantial transient expression of CXCL8 / IL8, vi) suppresses the expression of proliferation markers, such as CDK1, KI67 and T0P2A; and / or vii) does not negatively affect beta cell fraction, maturation and / or function.

[0232] In one embodiment, low-dose X-ray irradiation with between 0.8 - 10 Gy i) eliminates ductal cyst formation; ii) eliminates pluripotent cells in vitro, preferably by inducing growth arrest; iii) does not cause long-term damage to DNA; iv) does not induce a SASP phenotype in ILCs, such as expression of PAI-l / SERPIN El; v) does not induce substantial transient expression of CXCL8 / IL8, vi) suppresses the expression of proliferation markers, such as CDK1, KI67 and TOP2A; and / or vii) does not negatively affect beta cell maturation and / or function. viii)

[0233] In one embodiment, low-dose X-ray irradiation with between 0.8 - 4 Gy i) eliminates ductal cyst formation; ii) eliminates pluripotent cells in vitro, preferably by inducing growth arrest; iii) does not cause long-term damage to DNA; iv) does not induce a SASP phenotype in ILCs, such as expression of PAI-l / SERPIN El; v) does not induce substantial transient expression of CXCL8 / IL8, vi) suppresses the expression of proliferation markers, such as CDK1, KI67 and TOP2A; and / or vii) does not negatively affect beta cell maturation and / or function.

[0234] In one embodiment, low does X-ray irradiation with up to 10 Gy does not negatively affect cell composition. The fraction of mono-hormonal beta cells (NKX6.1 / C-peptide double-positive cells) and expression of key pancreatic and beta cell markers PDX1 and NKX6.1. X-ray irradiation, total endocrine cell fraction (CHGA+) or on the overall cell profile was not adversely affected. The ILCs mature into fully functional ILCs in vivo after transplantation.

[0235] In one embodiment, low-dose X-ray irradiation with less than 10 Gy does not negatively affect secretory capacity of end-stage PSC-derived beta-cell clusters.

[0236] In one embodiment, the irradiated PSC-derived beta-cell clusters remain devoid of unwanted cell growth in vivo for at least 1 year while maturing normally, and while retaining full function during the entire observation period. In one embodiment, the irradiated PSC-derived beta-cell clusters remain devoid of unwanted cell growth and retain full function in vivo for at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years.

[0237] Medical use of the invention

[0238] In another aspect, the invention also provides a pharmaceutical composition comprising the population of PSC-derived islet-like clusters (ILCs) of the invention or the cell transplant of the invention and one or more pharmaceutically acceptable excipients.

[0239] In another aspect, the invention provides the population of PSC-derived ILCs of the invention, the cell transplant of the invention or the pharmaceutical composition of the invention for use in the treatment.

[0240] In one embodiment, the invention provides the use of the population of PSC-derived ILCs of the invention, the cell transplant of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament.

[0241] In another aspect, the invention provides the population of PSC-derived ILCs of the invention, the cell transplant of the invention or the pharmaceutical composition of the invention for use in the treatment of diabetes.

[0242] In one embodiment, the invention provides the use of the population of PSC-derived ILCs of the invention, the cell transplant of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of diabetes. In one embodiment, the invention provides a method of treating diabetes, comprising administering the population of PSC-derived ILCs of the invention, the cell transplant of the invention or the pharmaceutical composition of the invention to a subject in need thereof.

[0243] In one embodiment, the treatment comprises transplantation of the in vitro irradiated PSC- derived ILCs in a subject suffering from diabetes. In one embodiment, the diabetes is type I diabetes. In other embodiments, the diabetes is another form of diabetes such as advanced type II diabetes, type III diabetes (T3D), diabetes after pancreatectomy e.g. due to recurrent pancreatitis or pancreatic tumors, forms of diabetes caused by genetic mutations, severe autoimmune diabetes (SAID), severe insulin-deficient diabetes (SIDD), or severe insulinresistant diabetes (SIRD).

[0244] In one embodiment, the treatment increases functional beta cell mass in subjects with diabetes. In one embodiment, the treatment reduces or eliminates hypoglycemic episodes and / or longterm diabetic complications in subjects with diabetes. In one embodiment, the treatment enables insulin independence in subjects with diabetes.

[0245] In one embodiment, the increase of functional beta cell mass is at least 10% compared to untreated subjects with diabetes. In one embodiment, the increase of functional beta cell mass is at least 50% compared to untreated subjects with diabetes. In one embodiment, the increase of functional beta cell mass fully restores beta cell function, preferably to the level required for fully normalized glycemic control in an individual.

[0246] Pharmaceutical compositions in the cell therapy field can be administered with cells or cell clusters (such as ILCs) suspended in media or buffers with pH, osmolarity and optionally cell survival promoting additives or supplements which are a) suitable to ensure survival and function of the administered cells and b) suitable and safe for administration to humans. Media, buffer and excipients should be non toxic and of suitable quality grade, for example, GMP or clinical grade.

[0247] Further Embodiments

[0248] The invention is also described by the following items: 1. A method of reducing and / or eliminating proliferative off-target cells in PSC-derived isletlike clusters (ILCs) by low-dose X-ray irradiation in vitro, comprising a) obtaining a population of cells comprising PSC-derived ILCs; and b) irradiating the population of cells with low-dose X-ray, wherein the low-dose is equal to or less than 4 Gy, preferably equal to or less than 2 Gy.

[0249] 2. The method according to item 1, wherein the unwanted cell growth arises from proliferative off-target cells selected from the group consisting of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells.

[0250] 3. An in vitro method of producing PSC-derived ILCs with improved safety profile suitable for cell therapy, the method comprising a) producing ILCs by culturing PSC under conditions suitable for the formation of ILCs; and b) irradiating the ILCs produced in step a) with low-dose X-ray, wherein the low-dose is equal to or less than 4 Gy, preferably equal to or less than 2 Gy.

[0251] 4. The method according to any one of the preceding items, wherein the method further comprises a step of c) culturing the population of PSC-derived ILCs after low-dose X-ray irradiation for at least 7 days; and / or d) monitoring whether the low-dose X-ray irradiated ILCs are free of proliferative off-target cells.

[0252] 5. The method of item 4, wherein monitoring whether the irradiated ILCs are free of proliferative off-target cells comprises determining the expression of one or more proliferation markers, wherein cells positive for one or more proliferation marker are reduced compared to controls that were not irradiated, preferably wherein the one or more proliferation markers are selected from the group consisting of CDK1, Ki67 and TOP2A.

[0253] 6. The method of item 4, wherein monitoring whether the irradiated ILCs are free of proliferative off-target cells comprises detection of proliferative cells, preferably using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, mesenchymal cell plating assay and ductal cell assay, or longterm in vivo studies in immunodeficient animals.

[0254] 7. A population of PSC-derived islet-like clusters (ILCs) suitable for cell therapy, obtained by irradiation with low-dose X-ray at a dose of equal to or less than 4 Gy, preferably equal to or less than 2 Gy, in vitro.

[0255] 8. A population of PSC-derived ILCs suitable for cell therapy obtained by the method of any one of items 1 to 6.

[0256] 9. A cell transplant comprising the population of PSC-derived ILCs of any one of items 7 or 8.

[0257] 10. A pharmaceutical composition comprising the population of PSC-derived ILCs of any one of items 7 or 8 or the cell transplant of item 9 and one or more pharmaceutically acceptable excipients.

[0258] 11. The population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9 or the pharmaceutical composition of item 10 for use in treatment.

[0259] 12. The population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9 or the pharmaceutical composition of item 10 for use in the treatment of diabetes.

[0260] 13. The population of PSC-derived ILCs, the cell transplant or the pharmaceutical composition for use of any one of items 11 or 12, wherein the treatment comprises transplantation of ILCs in a subject suffering from diabetes.

[0261] 14. The method of any one of items 1-6, the population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9, the pharmaceutical composition of item 10 or the use of any one of items 11 or 12, wherein the low-dose X-ray irradiation does not induce

[0262] (i) long-term DNA damage;

[0263] (ii) expression of the senescence markers, preferably selected from the group consisting of SERPINE1 / PALI, MMP1, CXCL1 and MMP3;

[0264] (iii) transient expression of CXCL8 and / or (iv) impairment of ILC cell maturation or function.

[0265] 15. The method, the population of PSC-derived ILCs, the population of PSC-derived ILCs, the cell implant, or the use of item 14, wherein long-term DNA damage is measured by determining the expression of pH2AX.

[0266] 16. The method, the population of PSC-derived ILCs, the population of PSC-derived ILCs, the cell implant, or the use of item 14, wherein ILC cell maturation is determined by determining the levels of one or more hormone markers selected from the group consisting of C-peptide, GCG, SST and 5-HT and / or one or more pancreatic cell markers selected from the group consisting of PDX1 and NKX6.1.

[0267] 17. The method, the population of PSC-derived ILCs, the population of PSC-derived ILCs, the cell implant, or the use of item 14, wherein ILC cell function is determined by measuring KCL induced insulin secretion in vitro; and / or by measuring overall cell yield; and / or by measuring percentage of beta-cells; compared to controls that were not irradiated.

[0268] 18. The method of any one of items 1-6, the population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9, the pharmaceutical composition of item 10 or the use of any one of items 11 or 12, wherein low-dose X-ray irradiation supresses the unwanted growth of ductal cysts, mesenchymal cell masses and / or residual pluripotent cell masses in the ILCs.

[0269] 19. The method of any one of items 1-6, the population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9, the pharmaceutical composition of item 10 or the use of any one of items 11 or 12, wherein the ILCs comprise a decreased percentage of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells; after low-dose X-ray irradiation compared to controls that were not irradiated.

[0270] 20. The method of any one of items 1-6, the population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9, the pharmaceutical composition of item 10 or the use of any one of items 11 or 12, wherein after low-dose X-ray irradiation the percentage of mitotic cells in the PSC-derived ILCs is less than 10%, preferably less than 1%.

[0271] 21. The method of any one of items 1-6, the population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9, the pharmaceutical composition of item 10 or the use of any one of items 11 or 12, wherein the low-dose X-ray irradiated ILCs of the invention form ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses at lower rates than controls that were not irradiated.

[0272] 22. The method of any one of items 1-6, the population of PSC-derived ILCs of any one of items 7 or 8, the cell transplant of item 9, the pharmaceutical composition of item 10 or the use of any one of items 11 or 12, wherein after low-dose X-ray irradiation, the ILCs do not form detectable ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, cell plating assay for pluripotent cell detection, ductal cyst formation and mesenchymal cell detection assay, in vivo transplantation into immunodeficient animals, or detection in patients (e.g. using imaging or sentinel devices).

[0273] 23. The population of PSC-derived ILCs, the cell transplant or the pharmaceutical composition for use of any one of items 11 or 12, wherein the ILCs do not form detectable ductal cysts, mesenchymal cell masses and / or pluripotent cell masses for at least 5 years after implantation; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, cell plating assay for pluripotent cell detection, ductal cyst formation and mesenchymal cell detection assay, in vivo transplantation into immunodeficient animals, or detection in patients (e.g. using imaging or sentinel devices).

[0274] 24. A population of PSC-derived islet-like clusters (ILCs) for use in treatment, wherein the ILCs have been irradiated with low-dose X-ray at a dose of equal to or less than 4 Gy, preferably equal to or less than 2 Gy, prior to treatment. 25. A population of PSC-derived islet-like clusters (ILCs) for use in the treatment of diabetes, wherein the ILCs have been irradiated with low-dose X-ray at a dose of equal to or less than 4 Gy, preferably equal to or less than 2 Gy, prior to treatment.

[0275] 26. A cell transplant comprising PSC-derived islet-like clusters (ILCs) for use in treatment, wherein the ILCs have been irradiated with low-dose X-ray at a dose of equal to or less than 4 Gy, preferably equal to or less than 2 Gy, prior to treatment.

[0276] 27. A cell transplant comprising PSC-derived islet-like clusters (ILCs) for use in the treatment of diabetes, wherein the ILCs have been irradiated with low-dose X-ray at a dose of equal to or less than 4 Gy, preferably equal to or less than 2 Gy, prior to treatment.

[0277] 28. The method of item 4, wherein a late intermediate stage (e.g. EN stage, day 16) during ILC manufacturing is cryopreserved, prior to thaw, finalization of differentiation and irradiation before harvesting of ILCs for transplantation.

[0278] 29. The method of item 4, wherein a late intermediate stage (e.g. EN stage, day 16) during ILC manufacturing is cryopreserved, and frozen ILCs are irradiated, returned to cryostorage, prior to thaw, finalization of differentiation before harvesting of ILCs for transplantation.

[0279] EXAMPLES

[0280] Technical aspects of X-ray irradiation

[0281] Irradiation data have been obtained using two different irradiation devices. To ensure comparability, the actual delivered doses were quantified within the respective cell containers (to exclude any differences induced e.g. by radiation absorption by lids, container walls or media) using calibrated dosimetry devices that can be placed inside and / or underneath the containers.

[0282] X-rays were chosen over y-rays for irradiation; this avoids hazards and cost associated with y- ray sources; X-rays are expected to be equally effective in terms of biological effects (e.g. cell cycle arrest or killing of proliferating cells). In some examples, low-dose X-ray irradiation was done using a Kubtec Xcell50 benchtop irradiation device.

[0283] Most irradiation data were obtained with a Precision X-ray MultiRad225. It features a more powerful X-ray source, enabling shorter irradiation pulses to deliver the same dose. This device is internally metered (it has a dosimetry unit below the material platform in the irradiation chamber), allowing tracking and documentation of proper irradiation dose delivery.

[0284] Data generated with the built-in dosimeter of the MultiRad225 and doses delivered with the Kubtex Xcell50 were cross-validated with small dosimetry chips or rods (e.g. myOSL chips, Radpro International GmbH, Remscheid, Germany or rod-shaped TL-detectors, Materialprufungsamt Nordrhein-Westfalen, Dortmund, Germany) placed inside the irradiation chamber and inside and / or below cell containers, fully validating that the delivered doses were in agreement with the expected doses for both devices, and regardless of container or media thickness.

[0285] Overall, it was ensured that radiation doses administered to cells with both devices were fully comparable and reproducible.

[0286] Example 1: Elimination of pluripotent iPSCs with low-dose X-ray irradiation iPSC clusters were generated from pluripotent TC-1133 cells in pluripotent culture medium. On the day of irradiation, a cluster sample was taken, dissociated and cell count quantified upon which clusters were moved to ultra-low attachment six well plates before the actual irradiation was performed. Plates were placed in the irradiation device and irradiation was performed with 0-0.8 Gy (XCell50, common setting 50 kV and 1 mA with no filtering). After irradiation, clusters were moved to a fresh ultra-low attachment plate and cultivation was continued in pluripotent cell medium for an additional six days.

[0287] When assessed after 6 days, the pluripotent cell cluster quantity and cell count were considerably decreased in the irradiated conditions, especially with doses higher than 0.2 Gy. Furthermore, irradiated iPSC clusters were architecturally less round and many clusters showed necrotic cores (Figure 1 A). After dissociation for single cell count, it was determined that cell loss was 97-99% compared to the untreated control group (Figure 1 B).

[0288] In parallel, clusters were dissociated after irradiation and seeded in 2D to allow for assessment of the ability to form typical iPSC colonies. Alkaline phosphatase (“ALPL”, a pluripotency marker) stainings were performed after 4 days, which was the timepoint when control / non- irradiated cells became fully confluent.

[0289] Consistent with cell counts of cells cultured in 3D, the cells that had been irradiated with doses of 0.4 Gy or more lost the ability to form typical colonies, although they retained positivity for ALPL. For the 0.8 Gy dose, only small residual cell clumps were observed on day four, which subsequently died completely (Figure 2).

[0290] These results show that X-ray irradiation with 0.8 Gy completely abrogates the ability of pluripotent iPSCs to survive and grow, even when cultured under optimal conditions in vitro (= in the presence of matrix and media optimized for pluripotent cell culture).

[0291] Example 2: Low-dose X-ray irradiation has no negative impact on differentiated ILC cell composition in day 20 ILCs, while it can eliminate proliferating spiked-in iPSC and other proliferating cells.

[0292] In a first experiment (TC-1133 3D194 and irradiation exp. 10) 5% iPSCs were spiked in as clusters into day-20 late-stage clusters, followed by immediate X-ray irradiation and a one-day incubation before cell composition was assessed by flow cytometry, and cells were then implanted into animals (described below in Example 10). Irradiation in a KubTec Xcell50 with 0.8 Gy, which was sufficient to fully kill or arrest pluripotent iPSCs (as shown in Figures 1 and 2) had no adverse effects on mono-hormonal cell fraction or cell number / survival (Figure 3). In a repeat experiment, day 20 ILCs were irradiated with 0, 0.4, 0.8 and 1.2 Gy in a Kubtec Xcell50, and cell composition was assessed by flow cytometry one day later. As shown in Figure 4, low-dose X-ray irradiation had no adverse effects on cell composition, with fractions of mono-hormonal beta cells (C-peptide / NKX6.1 double positive), CHGA-positve endocrine cells and NKX6.1 / PDX1 double positive cells remaining unchanged (Figure 4). Example 3: Low-dose irradiation has no effect on KCl-induced insulin secretion in day 21 ILCs

[0293] X-Ray irradiation did not negatively affect the secretory capacity of end-stage iPSC-derived beta-cell clusters in a secretion assay using low glucose vs. a low glucose plus high potassium chloride concentration. KCl-induced depolarization was used to trigger insulin secretion since the obtained ILC contained immature beta cells, which, while fully committed to the beta cell fate (insulin / C-peptide and NKX6.1 double positive, mono-hormonal), do not yet respond to high glucose challenges. Both basal and KCl-depolarization-triggered insulin secretion were not affected by X-ray irradiation with a dose of 0.8 Gy (Figure 5), demonstrating that low-dose X-ray irradiation does not impact beta cell function in vitro.

[0294] Example 4: Generation of day 23 end-stage ILCs for low-dose irradiation testing, and absence of irradiation effects on cluster integrity, cell composition or long-term DNA damage by day 23 low-dose X-ray irradiation

[0295] In a first experiment (Experiment MO 10), day 23 end-stage ILCs were generated by thawing late-stage intermediate ILC material cryopreserved at day 16, followed by thawing and finalization of differentiation until the equivalent of day 23. Irradiation was performed with fresh (non-frozen) ILCs in ultra-low attachment 6-well plates, in a MultiRad225 with doses of 0, 0.8 and 10 Gy, using the following device settings: Potential energy 225 kV, current: 17.8 mA, X-ray source distance 29.5 cm, dose rate: 7.7 Gy / min, X-ray filtration 2 mm Alu filter. Cluster

[0296] In another experiment (Exp. MO 13), day 23 ILCs were generated without intermittent cry opreservation. As for MO 10, irradiation was performed in 6-well plates on day 23, using X- ray doses of 0, 0.8, 4 and 10 Gy, in a Multirad225 with the same device settings as for MO 10 to assess effects of an extended X-ray dose range with regard to impact on cell survival, cell composition and eliminating unwanted cell growth, and to create a dose-response relationship over time. To this end, fresh (non-frozen) islet-like clusters (ILCs) were irradiated with a MultiRad225 with different doses at day 23 and analyzed at 2h, 24h and 48h. (n=2). Device settings were: Potential energy 225 kV, current: 17.8 mA, X-ray source distance 29.5 cm, dose rate: 7.7 Gy / min, X-ray filtration 2 mm Al filter.

[0297] No negative impact of X-ray irradiation with the doses 0.8 Gy, 4 Gy and 10 Gy on ILC integrity or appearance could be observed at the tested time points (Figures 6 and 8). No changes in viability after dissociation were observed up to 48h after irradiation at all X-ray doses (Figure 9).

[0298] Analysis of fresh ILCs irradiated with up to 10 Gy revealed no adverse effects on cell composition, indicated by an unchanged fraction of mono-hormonal beta cells (NKX6.1 / C- peptide double-positive cells) and unchanged expression of key pancreatic and beta cell markers PDX1 and NKX6.1. Low-dose X-ray irradiation also had no adverse effect on total endocrine cell fraction (CHGA+) or on the overall cell profile (Figure 7).

[0299] Example 5: Low-dose X-ray irradiation causes no long-term DNA damage

[0300] End-stage (d23) ILCs generated by directed differentiation, cry opreservation of intact clusters as EN-stage, thawing, and subsequent completion of differentiation were irradiated with 0, 0.8 and 10 Gy X-rays (Exp. MO10) as outlined in Example 4. In Exp. MO13, day 23 ILCs generated without intermittent cryopreservation were irradiated with 0, 0.8, 4 and 10 Gy as outlined in Example 4. Phosphorylated H2AX / gammaH2AX (pH2AX / yH2AX, described in Mah LJ, El- Osta A, Karagiannis TC. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010 Apr;24(4):679-86. PMID: 20130602.) was detected by flow-cytometry to assess X-ray-induced DNA damage. Results are shown in Figure 10 for MO10 (Fig. 10A) and MO I 3 (Fig. 10B).

[0301] Whereas ILCs irradiated with a dose of 0.8 Gy and 4 Gy showed only transient elevation of the pH2AX signal, the signal persisted in cells irradiated with 10 Gy after 24 hours, and even at 48 hours. This indicates that despite the fact that 0.8 Gy X-ray irradiation is able to eliminate ductal cyst formation, and to kill / arrest pluripotent cells in vitro, it does not cause long-term damage to DNA. The 4 Gy X-ray dose sample was also largely devoid of long-term DNA damage, showing that low-dose X-ray irradiation with doses in the 0.8-4 Gy range, and lower than 10 Gy are both effective and safe with regard to clearing / arresting of unwanted cells, ILC survival, function, and long-term DNA damage.

[0302] Example 6: Low-dose X-ray irradiation causes no induction of a broad senescence- associated secretory phenotype (SASP), and no / minimal elevation of SASP factor SERPIN El / PAI-1 Irradiation has been shown to cause a cellular senescence phenotype (Zhang et al. 2016).

[0303] Cellular senescence is associated with the Senescence-Associated Secretory Phenotype (SASP). Data on senescence in beta cells are mixed, with some articles claiming that SASP expression contributes to diabetes pathology, while others do not agree with this viewpoint (Lee et al. 2023; Thompson et al. 2019). In general, SASP induction in beta cells on a per cell basis is expected to be detrimental for beta cell survival and / or can contribute to attracting / activating beta cell killing immune cells.

[0304] Within the set of SASP markers tested (MMP1, SERPIN El / PAI-1, CXCL1, MMP3; analysis done by qRT-PCR), expression of MMP1, CXCL1, MMP3 was not or not strongly and consistently activated by irradiation in Experiments MO 10 and MO 13 at all irradiation doses and time points tested (Figure 11 shows representative data from Exp. MO 13).

[0305] However, SERPIN El / PALI was elevated after X-ray irradiation of end-stage ILCs with a dose of 10 Gy, but not with a dose 0.8 Gy (Exp. MO10, Figure 12A). A minimal elevation of SERPIN El expression was seen at 4 Gy, but at a substantially lower level compared to cells irradiated at 10 Gy (Exp. MO13, Figure 12B). SERPIN El / PALI is a key marker and mediator of replicative senescence; it has been linked to fibrotic changes, and SASP -mediated immune cell activation (Zhang et al. 2021).

[0306] Overall the results show that X-ray irradiation with less than 4 or 10 Gy does not induce expression of PALI / SERPIN Eland a SASP phenotype in ILCs, showing that it is suitable for the treatment of a diabetes cell therapy product. Irradiation with 4 Gy induced only a minimal increase in SERPIN El expression, substantially lower compared to the effect seen with 10 Gy.

[0307] Example 7: Low-dose X-ray irradiation causes no induction of CXCL8 / IL8 expression

[0308] Irradiation with high doses of X-rays has been shown to induce expression and secretion of certain innate and inflammatory cytokines, also as a consequence of release of mitochondrial DNA into the cytoplasm (Tigano M, Vargas DC, Tremblay-Belzile S, Fu Y, Sfeir A. Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance. Nature. 2021 Mar;591(7850):477-481. PMID: 33627873.). Such an effect would be highly detrimental in ILC preparations, because it could negatively affect pre- and / or post-transplantation ILC survival and function, e.g by attracting immune cells, and / or by stimulating inflammatory factor secretion and killing mediated by these cells. To determine the effects of different doses of X-ray irradiation on ILCs, qPCR analysis the expression of inflammatory cytokines IFNG, IFNB1 and CXCL8 / IL8 was performed with day 23 ILCs, irradiated with 0, 0.8, 4 and 10 Gy, at 2, 24 and 48 hours post irradiation (Exp. MO13). As shown in Figure 13, expression of CXCL8, a cytokine known to attract and activate especially neutrophils and myeloid cells, was strongly and significantly upregulated in a transient manner at the 10 Gy dose but not below. IFNG and IFNB1 were not induced in all tested irradiation doses and time points. This shows that unlike high dose irradiation with 10 Gy, low-dose irradiation does not induce a transient expression of the inflammatory and immune cell attracting cytokine CXCL8 / IL8. This makes low-dose irradiation especially suitable for ILCs that are transplanted within 24 hours after irradiation.

[0309] Example 8: Low-dose irradiation strongly and persistently suppresses the expression of proliferation markers

[0310] The effect of low-dose X-ray irradiation on the expression of key proliferation markers CDK1, KI67 and TOP2A was determined at different timepoints after irradiation with ILCs from Experiments MO 11 and MO 13.

[0311] It was found that low-dose X-ray irradiation suppresses the expression of proliferation markers CDK1, KI67 and TOP2A in irradiated end-stage / d23 ILCs. Data from Experiment MO10 are shown in Figure 14A, demonstrating that already a dose of 0.8 Gy caused a strong suppression of proliferation marker expression 24 hours post irradiation, and therefore a suppression of unwanted cell growth potential in the irradiated ILC preparations. Increasing the dose from 0.8 Gy to 10 Gy caused a further increase in suppression; however, this increase was smaller compared to the drop in expression seen already when going from 0 Gy to 0.8 Gy.

[0312] These results were confirmed and extended by data from Experiment MO 13, in which an additional dose (4 Gy) and time point (48 hrs) was included. As shown in Figure 15 A, the suppression of proliferation marker expression not only persisted at the 48 hour time point, but also became more pronounced.

[0313] With 4 Gy irradiation, suppression of proliferation marker expression reached a plateau, and there was no additional benefit from increasing the dose further to 10 Gy. A near maximal effect was already apparent at 0.8 Gy. This means that with 0.8 and 4 Gy, while there is either no or only limited DNA damage persisting at 24 and 48 hours (as shown by pH2AX flow cytometry analysis, see above), suppression of proliferation was already near maximal or maximal, and persisted with no sign of waning.

[0314] The ideal range for irradiation appeared to be between 0.8 Gy and 4 Gy, and below 10 Gy in end-stage differentiation ILCs.

[0315] Example 9: Low-dose X-ray irradiation triggers a p53-driven positive feedback loop suppressing proliferation

[0316] The CDK inhibitor p21 / CDKNl A is a key mediator of irradiation-induced growth arrest (Kreis NN, Louwen F, Yuan J. The Multifaceted p21 (Cipl / Wafl / CDKNIA) in Cell Differentiation, Migration and Cancer Therapy. Cancers (Basel). 2019 Aug 21; 11(9): 1220. PMID: 31438587). The long non-coding RNA DINO has been shown to be under transcriptional control of p53 (with p53 being an activator of DINO expression), but to also enhance p53 function via a direct binding mechanism, leading to the formation of a positive feedback loop (Schmitt et al., An inducible long noncoding RNA amplifies DNA damage signaling. Nat Genet. 2016 Nov;48(l l): 1370-1376. PMID: 27668660). Expression of CDKN1A and DINO was analysed by qPCR in irradiated end-stage ILCs to gain additional insights in the molecular mechanisms of growth arrest effects of low-dose X-ray irradiation.

[0317] Data from Experiment MOI 1 and MO13 showed that irradiation with an X-ray dose of 0.8 Gy was sufficient to induce a substantial increase in the expression of both CDKN1A and DINO. Increasing the X-ray dose further to 4 Gy in MO 13 resulted in a further increase, with a maximal effect seen in ILCs irradiated with 10 Gy. Elevated CDKN1A and DINO expression persisted at 48 hours with 0.8 and 4 Gy, despite the fact that at this point, no DNA damage was detectable by pH2AX staining (MO11, Figures 14B and C and MO13, Figures 15B and C). Overall, the data are in agreement with low-dose X-ray irradiation triggering a substantial and persistent growth arrest, which in turn is in part or wholly due to a positive feedback loop involving p53 activation, DINO and CDKN1 A. A schematic representation of this feedback loop can be found in Figure 16.

[0318] Example 10: Low-dose X-ray irradiation of end-stage differentiation ILCs suppresses unwanted cell growth in a long-term (1 year) in vivo study without negative impacts on beta cell maturation or function To determine the effects of low-dose X-ray irradiation on unwanted cell growth from transplanted ILCs in an in vivo study, end-stage ILCs (day 22 of differentiation) were irradiated either with 0 Gy or with 0.8 Gy, and subsequently transplanted under the kidney capsule of immunodeficient mice in which endogenous beta cells had been ablated with streptozotocin. The control group with non-irradiated cells received a graft containing 1.5 million beta cells, while the 0.8 Gy group received a dose containing 1.3 million beta cells. Subsequently, random fed glucose levels were determined on a regular basis. Oral glucose tolerance tests and quantification of circulating human C-peptide were performed at the indicated time points.

[0319] The implanted populations contained a high fraction of mono-hormonal beta cells (61 and 59%, respectively), a low fraction of Ki67+ cells, and consisted almost exclusively of pancreatic (PDX1 -positive) cells (data obtained by flow cytometry).

[0320] Since the implanted ILCs contained immature / fetal-stage beta cells, beta cell maturation to acquire full glucose responsiveness and insulin content occurs after implantation, resulting in steadily improving random fed blood glucose levels until a steady state at the human setpoint is reached. A steady state at the human setpoint below lOOmg / dl confirms that glycemic control is mediated entirely by implanted human cells. As can be seen in Figure 17A, irradiated ILCs matured at the same rate as non-irradiated cells, and also achieved full glucose normalization at the human glucose setpoint, with no significant difference relative to non-irradiated control material.

[0321] To obtain more information on the performance of the implanted ILCs, circulating human C- peptide were determined in blood samples taken 2, 6 and 12 weeks post implantation.

[0322] Levels of human C-peptide increased over time as expected due to the maturation of beta cells contained in the ILCs. As for random fed blood glucose levels, there was no difference between irradiated vs. non-irradiated ILCs in the amount of human C-peptide secreted into the circulation of recipient mice (Figure 17B) .

[0323] To determine the response of implanted ILCs to an oral glucose bolus, oral glucose tolerance tests (oGTTs) were performed at different time points during the experiment. Results are shown in Figure 18. As expected, performance of control animals in the oGTT improved over time, with only minimal glucose excursion and rapid clearing of the ingested glucose at 32 weeks. As for the previous tested parameters, there was no difference in oGTT performance between nonirradiated and irradiated cells.

[0324] At the end of the in-life observation period of 365 days, animals were sacrificed, and graftcontaining kidneys were initially assessed macroscopically. Graft-containing kidneys were subsequently processed for histology. Upon explantation, all grafts were visible as whitish, smooth areas under the kidney capsule. None of the animals had macroscopically visible aberrations such as cysts or uncontrolled growth in the graft.

[0325] A subset of animals in both groups had to be sacrificed before the planned end of the study. This was due either to infections (to which the immunodeficient recipient mouse strain NXG is more susceptible than wild type mice) or lymphoma (confirmed by a trained pathologist, an occasional finding in NSG and NXG mice, Foreman et al. 2011; Finesso et al. 2023). Photographs of graft-containing kidneys from animals of the non-irradiated control group are shown in Figure 19, graft-containing kidneys from recipients of irradiated ILCs are shown in Figure 20. If kidneys are from animals dissected before the planned end of the study, the day of dissection is indicated.

[0326] Histological analysis revealed well-organized grafts consisting entirely or mainly (see below) of endocrine cells (see below), including the expected (based on implant cell composition) high fraction of C-peptide positive beta cells in all animals.

[0327] An overview of grafts (hormone stain, combining C-peptide, GCG, SST and 5-HT) combined with DAPI, as well as a human mitochondria-specific stain to distinguish human graft cells from mouse host tissue) is shown in Figures 21 and 22 for control animals. Graft overviews from recipients of irradiated ILC grafts are shown in Figure 23.

[0328] Grafts were also analyzed for the presence of unwanted cell growth using a combination of H&E staining and assessment by a trained pathologist. No signs of teratoma or mesenchymal cell masses were detectable in either the control or the irradiated groups.

[0329] For CK19-positive ductal cysts, detailed histological analysis (CK19-staining on sections spaced every 170pm through the entire graft in all animals) revealed the presence of one ductal cyst each in two control group animals. No CK19 positive cysts were detectable in the X-ray group. Occasional single CK19 positive cells were observed in animals from both groups but were judged to be not problematic.

[0330] An example of a ductal cyst detected in the non-irradiated control group as well as an overview of the number of analyzed sections vs. ductal cysts identified is provided in Figure 24. Overall, while the tendency to form unwanted cell growth, including ductal cysts, was already very low in the ILC material that was transplanted in this experiment, low-dose X-ray irradiation was able to reduce ductal cyst formation to zero, while leaving beta cell maturation and long-term function fully intact, and without inducing any de novo tumor formation e.g. due to radiation- induced DNA damage.

[0331] Example 11: Low-dose X-ray irradiation at a late intermediate stage of differentiation does not impact in vitro differentiation, in vivo maturation, or long-term function in vivo

[0332] PSC-derived immature beta cells, similar to human fetal beta cells, can complete maturation upon implantation into animals or patients, and thereby become glucose responsive and also achieve an overall secretory output and response profile highly similar to isolated human donor islets. Function of implanted PSC-derived beta cells can be assessed e.g. in recipient mice by analyzing circulating human C-peptide levels, with C-peptide being a co-secreted by-product of insulin synthesis for which species-specific assays are available. The same readout is also used to assess beta cell function in humans.

[0333] To determine the ability of low-dose X-ray irradiation to prevent unwanted cell growth either from residual pluripotent iPSCs, ductal or mesenchymal cells, an in vivo study was conducted (TC-1133 3D217, X-Ray exp. 16). Based on data obtained previously, formation of teratomas was not expected with the manufacturing / differentiation process that was used to generate the ILCs for the study (W02023203208A1). In an attempt to enable, in parallel, the generation of additional data on low-dose X-ray effects on in vivo teratoma formation, a 1% spike-in of pluripotent iPSC clusters was performed at day 16 (EN-stage). However, no teratomas were observed upon implantation, showing that spiked-in pluripotent cells lost the ability to form tumors during maturation, presumably due to the maturation media. Irradiation with 0, 0.4 and 0.8 Gy X-rays was performed immediately after spike-in of pluripotent clusters, and irradiated clusters were matured in six well plates in vitro until day 23 (EN+7). Flow cytometry and other assessments were performed on that day, enabling normalization of transplanted cell dose on number of beta cells, and followed by implantation into non-diabetic immunodeficient mice. Clusters from the above experiment were implanted at day 23 under the kidney capsule of nondiabetic immunodeficient mice to assess the appearance of different types of unwanted cell growth in a timeframe of 3 months (EV023006). The implanted cell quantity was 1 million beta-cells (NKX6.1 / C-peptide double positive cells) per animal, corresponding to about 2.5 million total cells per animal. Due to the non-diabetic status of the recipient animals, only subtle effects on random fed blood glucose were expected during the duration of the study. However, beta cell maturation and function of implanted ILCs is expected to proceed in a similar fashion as in diabetic recipients, meaning that beta cell maturation and function can be assessed in irradiated vs. non-irradiated implants e.g. by using the quantification of circulating human C- peptide levels.

[0334] Circulating human C-peptide levels are shown in Figure 25. Substantial levels of circulating human C-peptide was detectable at all tested timepoints with increasing levels over time as expected. Importantly, no differences were observed between the treatment groups, indicating that irradiation had no negative impact on the maturation of beta cells and their function in vivo.

[0335] Example 12: Low-dose X-ray irradiation at differentiation day 16 (during endocrine differentiation) dose-dependently suppresses growth of ductal cysts and PDXl-negative cell masses

[0336] After 3 months, animals in study EV023006 were sacrificed, and kidneys containing ILC grafts were explanted. In a first step, an assessment of graft appearance was made, and macroscopic alterations leading to a deviation from the expected smooth whitish graft area presentation were noted.

[0337] Figures 26, 27 and 28 show macroscopic images of kidneys from the 0, 0.4 and 0.8 Gy groups, respectively. While non-irradiated animals showed numerous cystic and mesenchymal-like outgrowths, a reduced incidence was apparent in the 0.4 Gy group, while the 0.8 Gy group was devoid of any macroscopically visible graft alteration.

[0338] Grafts were then subjected to histological analysis. Sections were stained with hematoxylin / eosin (HE), and via immunofluorescence for CK19 (ductal cells), NKX6.1 / PDX1 (pancreatic cells), for human mitochondria to determine the extent of the graft, and in some cases for Ki67 as proliferation marker. Sections were examined by a trained pathologist. Histological data for control animals are shown on Figures 29-34. Histology data for recipient animals with 0.8 Gy-irradiated ILC grafts are shown in Figures 35-40. Histological analysis of grafts showed the absence of teratoma formation in all animals. Furthermore, it showed the presence of CK19-positive ductal cysts in control and to some extent in 0.4 Gy animals, but not in animals whose grafts had been irradiated with 0.8 Gy. Furthermore, PDX1 -negative, nonpancreatic (putative mesenchymal) cell masses were observed in the control and to a lesser extent in the 0.4 Gy groups, but not in the 0.8 Gy group.

[0339] Overall, irradiation of ILCs at a late intermediate differentiation stage (day 16) with 0.4 Gy partially and with 0.8 Gy X-ray irradiation completely prevented the formation of large ductal cystic outgrowths and also the formation of PDX1 -negative cell masses, without affecting in vivo beta cell maturation and function. Irradiation was safe, and there was no de-novo tumor formation. Low-dose X-ray irradiation was able to suppress unwanted cell growth although in this experiment, an ILC batch with relatively high propensity to form unwanted cell growth was used.

[0340] Example 13: The effect of low-dose X-ray irradiation on unwanted cell growth in ILCs can be demonstrated in an in vitro assay

[0341] To determine whether ductal cyst and other unwanted cell growth (e.g. mesenchymal / PDXl- negative cell masses) can be detected also without long-term in vivo studies, an in vitro assay was set up using Matrigel embedding of non-dissociated end-stage ILCs, followed by detection of ductal cysts by microscopy, and detection of a range of unwanted cell types using qPCR. For the assay, intact (non-dissociated) ILCs were embedded in a Matrigel layer, in a differentiation medium that enables ductal cyst formation, modified from Breunig et al, 2021.

[0342] Matrigel is a cell-line derived extracellular matrix mix consisting of approximately 60% laminin, 30% collagen IV and 8% entactin (Corning® Matrigel® Matrix FAQs). Other potentially useful matrices for a combined ductal cyst and mesenchymal cell growth assay can be generated e.g. from recombinant extracellular matrix proteins and growth factors.

[0343] ILCs were generated using the protocol outlines in (W02023203208A1), but omitting the use of gamma secretase-inhibitor to deliberately increase the number of non-endocrine committed cells in the end stage population, yielding ductal / progeni tor-enriched ILCs (dpILCs). Non- frozen dpILCs were irradiated in 6-well plates (ultra-low attachment), with the following irradiation parameters in a MultiRad225:

[0344] Doses: 0, 0.8, 4 and 10 Gy

[0345] Potential energy: 225 kV

[0346] Current: 17.8 mA

[0347] X-ray source distance: 29.5 cm

[0348] Dose / rate: 7.7 Gy / min

[0349] X-ray filtration: 2 mm Aluminum filter

[0350] The unwanted cell growth assay was performed in 6 well plates. Images of embedded clusters were taken daily until day 23+8, and qPCR data were acquired on day 23+8. For the last 24 hours, swelling of ductal cysts was enhanced by adding forskolin and IBMX, two cAMP signaling enhancers.

[0351] As can be seen in Figure 41, some expansion of cystic structures could be observed in all conditions during the initial days of incubation. However, in dpILC samples irradiated with 4 Gy or higher, cystic structures disappeared and were not re-established during the observation period. After cAMP-enhancer mediated expansion of cysts, differences between groups became more obvious, with no ductal cysts visible for the 4 and 10 Gy groups, while some cysts were visible with 0.8 Gy and even more so in the non-irradiated control. A high magnification representation of the assay outcome is shown in Figure 42, a low-magnification overview is shown in Figure 43.

[0352] Results are in line with the in vivo results presented above in that low-dose X-ray irradiation with less than 10 Gy is sufficient to abrogate unwanted cell growth from ILCs. The fact that in this in vitro experiment 0.8 Gy was not fully sufficient to abrogate all cystic outgrowth can be explained by the use of dpILCs generated without the use of endocrine-inducing gamma- secretase inhibitor, and therefore a very high fraction of multipotent pancreatic progenitors and potentially primitive ductal precursors.

[0353] Example 14: Low-dose X-ray irradiation of cryopreserved EN-stage ILCs has no adverse effects on cell composition

[0354] To determine whether low-dose X-ray irradiation could also be applied to cryopreserved late intermediate stage (day 16) ILCs, such ILCs were irradiated with 0, 0.5, 0.8, 1.0 and 10.0 Gy X-rays, using a MultiRad225 with settings described in Example 11. After removing cryovials from storage in liquid N2 they were placed in thin layers of thermal insulation (Styrofoam) before administering the short irradiation pulses (approximately 3.9 seconds for 0.5 Gy, 6.2 seconds for 0.8 Gy, .7.7 seconds for 1 Gy and 77 seconds for 10 Gy). After irradiation, frozen ILCs cells were returned immediately to cryostorage. Subsequently, ILCs were thawed, and endocrine differentiation was completed until the equivalent of day 23.

[0355] Assessment of ILC morphology at different stages during finalization of differentiation showed the presence of well defined clusters at all time points and for all doses (Fig. 44). However, while cell recovery was between 6 and 12 % and cell viability was high for control, 0.5 Gy, 0.8 Gy and 1 Gy irradiated cells, irradiation with 10 Gy induced strong cell death at the end of differentiation, so that only a small number of ILCs corresponding to a recovery of 0.5% remained on the equivalent of day 16+7 / 23 (Figure 45 A and Figure 45B).

[0356] Flow cytometry analysis at the end of differentiation revealed that while low-dose X-ray irradiation with 0.5, 0.8 or 1 Gy had no adverse effects on cell composition (shown by the absence of effects on the percentage of mono-hormonal beta cells and ISL1 -positive cells), irradiation of cryopreserved day 16 ILC material with 10 Gy resulted in a strongly decreased beta cell fraction on day 23 (Figure 46). The results demonstrate that cryopreserved, intact day 16 ILCs can be irradiation with X-ray irradiation less than 10 Gy without adverse effects on cell recovery, and beta cell fraction / cell composition. The combination of low irradiation dose with high dose rate provided by the MultiRad225 and comparable lab-scale X-ray devices enable rapid irradiation without thawing or undue warming of cryopreserved ILC material, and can be easily integrated into any GMP manufacturing workflow for ILCs.

[0357] References d'Adda di Fagagna F. Living on a break: cellular senescence as a DNA-damage response. Nat Rev Cancer. 2008 Jul;8(7):512-22. doi: 10.1038 / nrc2440. PMID: 18574463.; Ghosh AK, Vaughan DE. PALI in tissue fibrosis. J Cell Physiol. 2012 Feb;227(2):493-507. doi: 10.1002 / jcp.22783. PMID: 21465481; PMCID: PMC3204398.

[0358] Augsornworawat P, Maxwell KG, Velazco-Cruz L, Millman JR. Single-Cell Transcriptome Profiling Reveals P Cell Maturation in Stem Cell-Derived Islets after Transplantation. Cell Rep. 2020 Aug 25;32(8): 108067. PMID: 32846125; PMCID: PMC7491368.

[0359] Benomar K, Chetboun M, Espiard S, Jannin A, Le Mapihan K, Gmyr V, Caiazzo R, Torres F, Raverdy V, Bonner C, D'Herbomez M, Pigny P, Noel C, Kerr-Conte J, Pattou F, Vantyghem MC. Purity of islet preparations and 5-year metabolic outcome of allogenic islet transplantation. Am J Transplant. 2018 Apr;18(4):945-951. doi: 10.1111 / ajt.14514. Epub 2017 Nov 11. PMID: 28941330. Breunig M, Merkle J, Melzer M, Heller S, Seufferlein T, Meier M, Hohwieler M, Kleger A, Differentiation of human pluripotent stem cells into pancreatic duct-like organoids, STAR Protocols, Volume 2, Issue 4, 2021.

[0360] Chandra V, Ibrahim H, Halliez C, Prasad RB, Vecchio F, Dwivedi OP, Kvist J, Balboa D, Saarimaki-Vire J, Montaser H, Barsby T, Lithovius V, Artner I, Gopalakrishnan S, Groop L, Mallone R, Eizirik DL, Otonkoski T. The type 1 diabetes gene TYK2 regulates P-cell development and its responses to interferon-a. Nat Commun. 2022 Oct 26;13(1):6363. PMID: 36289205.

[0361] Finesso G, Willis E, Tarrant JC, Lanza M, Sprengers J, Verrelle J, Banerjee E, Hermans E, Assenmacher CA, Radaelli E. Spontaneous early-onset neurodegeneration in the brainstem and spinal cord of NSG, NOG, and NXG mice. Vet Pathol. 2023 May;60(3):374-383. PMID: 36727841; PMCID: PMC10150263.

[0362] Foreman O, Kavirayani AM, Griffey SM, Reader R, Shultz LD. Opportunistic bacterial infections in breeding colonies of the NSG mouse strain. Vet Pathol. 2011 Mar;48(2):495-9. PMID: 20817888; PMCID: PMC3101569.

[0363] Gubitosi-Klug RA, Braffett BH, Hitt S, Arends V, Uschner D, Jones K, Diminick L, Karger AB, Paterson AD, Roshandel D, Marcovina S, Lachin JM, Steffes M, Palmer JP; DCCTZEDIC Research Group. Residual P cell function in long-term type 1 diabetes associates with reduced incidence of hypoglycemia. J Clin Invest. 2021 Feb 1; 13 l(3):el43011. PMID: 33529168.

[0364] Hiyoshi H, Sakuma K, Asano S, Napier SC, Konagaya S, Mochida T, Ueno H, Watanabe T, Kassai Y, Matsumoto H, Ito R, Toyoda T. Identification and removal of unexpected proliferative off-target cells emerging after iPSC-derived pancreatic islet cell implantation. Proc Natl Acad Sci U S A. 2024 Apr 16; 121(16):e2320883121.

[0365] Hogrebe NJ, Ishahak M, Millman JR. Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes. Cell Stem Cell. 2023 May 4;30(5):530-548. PMID: 37146579.

[0366] Jennings RE, Berry AA, Strutt JP, Gerrard DT, Hanley NA. Human pancreas development. Development. 2015 Sep 15;142(18):3126-37. PMID: 26395141.

[0367] Nathan DM. Realising the long-term promise of insulin therapy: the DCCTZEDIC study. Diabetologia. 2021 May;64(5): 1049-1058. PMID: 33550441.

[0368] Nguyen HQ, To NH, Zadigue P, Kerbrat S, De La Taille A, Le Gouvello S, Belkacemi Y. Ionizing radiation-induced cellular senescence promotes tissue fibrosis after radiotherapy. A review. Crit Rev Oncol Hematol. 2018 Sep;129: 13-26. doi: 10.1016 / j.critrevonc.2018.06.012. Epub 2018 Jun 22. PMID: 30097231. Nostro MC, Keller G. Generation of beta cells from human pluripotent stem cells: Potential for regenerative medicine. Semin Cell Dev Biol. 2012 Aug;23(6):701-10. PMID: 22750147.

[0369] Mah LJ, El-Osta A, Karagiannis TC. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia. 2010 Apr;24(4):679-86. PMID: 20130602.

[0370] Meier JJ, Butler AE, Saisho Y, Monchamp T, Galasso R, Bhushan A, Rizza RA, Butler PC. Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans. Diabetes. 2008 Jun;57(6): 1584-94. doi: 10.2337 / db07-1369. PMID: 18334605.

[0371] Lee et al., Stress-induced P cell early senescence confers protection against type 1 diabetes by enabling the removal of senescent beta cells. Cell Metab. 2023 Dec 5;35(12):2200-2215.e9. PMID: 37949065.

[0372] Pagliuca FW, Millman JR, Gurtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. Generation of functional human pancreatic P cells in vitro. Cell. 2014 Oct 9;159(2):428-39. PMID: 25303535.

[0373] Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A, O'Dwyer S, Quiskamp N, Mojibian M, Albrecht T, Yang YH, Johnson JD, Kieffer TJ. Reversal of diabetes with insulinproducing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. 2014 Nov;32(l l): 1121-33. PMID: 25211370.

[0374] Rickels MR, Robertson RP. Pancreatic Islet Transplantation in Humans: Recent Progress and Future Directions. Endocr Rev. 2019 Apr l;40(2):631-668. PMID: 30541144.

[0375] Steffes MW, Sibley S, Jackson M, Thomas W. Beta-cell function and the development of diabetes-related complications in the diabetes control and complications trial. Diabetes Care. 2003 Mar;26(3):832-6. PMID: 12610045.

[0376] Thompson, Bhushan et al., Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes. Cell Metab. 2019 May 7;29(5): 1045-1060.el0. PMID: 30799288.

[0377] Zhang et al., Both Complexity and Location of DNA Damage Contribute to Cellular Senescence Induced by Ionizing Radiation. PLoS One. 2016 May 17;1 l(5):e0155725PMID: 27187621; Limbad et al., Astrocyte senescence promotes glutamate toxicity in cortical neurons. PLoS One. 2020 Jan 16;15(l):e0227887. PMID: 31945125.

[0378] Zhang et al., Hepatic stellate cell senescence in liver fibrosis: Characteristics, mechanisms and perspectives. Meeh Ageing Dev. 2021 Oct;199: 111572. PMID: 34536446.; Valentijn et al., Cellular senescence in the aging and diseased kidney. J Cell Commun Signal. 2018 Mar;12(l):69-82. PMID: 29260442.

Claims

CLAIMS1. A method of reducing and / or eliminating proliferative off-target cells in PSC-derived isletlike clusters (ILCs) by low-dose X-ray irradiation in vitro, comprising a) obtaining a population of cells comprising PSC-derived ILCs; and b) irradiating the population of cells with low-dose X-ray, wherein the low-dose is equal to or less than 10 Gy.

2. The method according to claim 1, wherein the unwanted cell growth arises from proliferative off-target cells selected from the group consisting of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells.

3. An in vitro method of producing PSC-derived ILCs with improved safety profile suitable for cell therapy, the method comprising a) producing ILCs by culturing PSC under conditions suitable for the formation of ILCs; and b) irradiating the ILCs produced in step a) with low-dose X-ray, wherein the low-dose is equal to or less than 10 Gy.

4. The method according to any one of the preceding claims, wherein the method further comprises a step of c) culturing the population of PSC-derived ILCs after low-dose X-ray irradiation for at least 7 days; and / or d) monitoring whether the low-dose X-ray irradiated ILCs are free of proliferative off-target cells.

5. The method of claim 4, wherein monitoring whether the irradiated ILCs are free of proliferative off-target cells comprises determining the expression of one or more proliferation markers, wherein cells positive for one or more proliferation marker are reduced compared to controls that were not irradiated, preferably wherein the one or more proliferation markers are selected from the group consisting of CK19, Ki67 and TOP2A.

6. The method of claim 4, wherein monitoring whether the irradiated ILCs are free of proliferative off-target cells comprises detection of unwanted proliferative cells, preferably using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, ductal cyst formation and mesenchymal cell detection assay, or transplantation into immunodeficient animals.

7. A population of PSC-derived islet-like clusters (ILCs) suitable for cell therapy, obtained by irradiation with low-dose X-ray at a dose of equal to or less than 10 Gy in vitro.

8. A population of PSC-derived ILCs suitable for cell therapy obtained by the method of any one of claims 1 to 6.

9. A cell transplant comprising the population of PSC-derived ILCs of any one of claims 7 or 8.

10. A pharmaceutical composition comprising the population of PSC-derived ILCs of any one of claims 7 or 8 or the cell transplant of claim 9 and one or more pharmaceutically acceptable excipients.

11. The population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9 or the pharmaceutical composition of claim 10 for use in treatment.

12. The population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9 or the pharmaceutical composition of claim 10 for use in the treatment of diabetes.

13. The population of PSC-derived ILCs, the cell transplant or the pharmaceutical composition for use of any one of claims 11 or 12, wherein the treatment comprises transplantation of ILCs in a subject suffering from diabetes.

14. The method of any one of claims 1-6, the population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9, the pharmaceutical composition of claim 10 or the use of any one of claims 11 or 12, wherein the low-dose X-ray irradiation does not induce(i) long-term DNA damage;(ii) expression of senescence markers, preferably selected from the group consisting of SERPINE1 / PALI, MMP1, CXCL1 and MMP3; and / or(iii) impairment of ILC cell maturation or function.

15. The method, the population of PSC-derived ILCs, the population of PSC-derived ILCs, the cell implant, or the use of claim 14, wherein long-term DNA damage is measured by determining the expression of pH2AX.

16. The method, the population of PSC-derived ILCs, the population of PSC-derived ILCs, the cell implant, or the use of claim 14, wherein ILC cell maturation is determined by determining the levels of one or more hormone markers selected from the group consisting of C-peptide, GCG, SST and 5-HT and / or one or more pancreatic cell markers selected from the group consisting of PDX1 and NKX6.1.

17. The method, the population of PSC-derived ILCs, the population of PSC-derived ILCs, the cell implant, or the use of claim 14, wherein ILC cell function is determined by measuring KCL induced insulin secretion in vitro; and / or by measuring overall cell yield; and / or by measuring percentage of beta-cells; compared to controls that were not irradiated.

18. The method of any one of claims 1-6, the population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9, the pharmaceutical composition of claim 10 or the use of any one of claims 11 or 12, wherein low-dose X-ray irradiation supresses the unwanted growth of ductal cysts, mesenchymal cell masses and / or residual pluripotent cell masses in the ILCs.

19. The method of any one of claims 1-6, the population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9, the pharmaceutical composition of claim 10 or the use of any one of claims 11 or 12, wherein the ILCs comprise a decreased percentage of i) ductal cells and / or proliferating multipotent pancreatic progenitors and cells derived thereof; ii) PDX1 -negative cells such as mesenchymal cells, and / or iii) residual pluripotent cells; after low-dose X-ray irradiation compared to controls that were not irradiated.

20. The method of any one of claims 1-6, the population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9, the pharmaceutical composition of claim 10 or the use of any one of claims 11 or 12, wherein after low-dose X-ray irradiation the percentage of mitotic cells in the PSC-derived ILCs is less than 10%, preferably less than 1%.

21. The method of any one of claims 1-6, the population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9, the pharmaceutical composition of claim 10 or the use of any one of claims 11 or 12, wherein the low-dose X-ray irradiated ILCs of the invention form ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses at lower rates than controls that were not irradiated.

22. The method of any one of claims 1-6, the population of PSC-derived ILCs of any one of claims 7 or 8, the cell transplant of claim 9, the pharmaceutical composition of claim 10 or the use of any one of claims 11 or 12, wherein after low-dose X-ray irradiation, the ILCs do not form detectable ductal cysts, mesenchymal cell masses, and / or pluripotent cell masses; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, ductal cyst formation and mesenchymal cell detection assay, or transplantation into immunodeficient animals.

23. The population of PSC-derived ILCs, the cell transplant or the pharmaceutical composition for use of any one of claims 11 or 12, wherein the ILCs do not form detectable ductal cysts, mesenchymal cell masses and / or pluripotent cell masses for at least 5 years after implantation; preferably wherein ductal cysts, mesenchymal cell masses and / or pluripotent cell masses are detected using an assay selected from the group consisting of BrdU proliferation assay, EdU proliferation assays, pluripotent cell plating assay, ductal cyst formation and mesenchymal cell detection assay, or transplantation into immunodeficient animals

Images