Composition and method for spreading insulin-producing islet cells and therapeutic use thereof

Abstract

The present invention discloses a composition and method for differentiating pancreatic cells into CD133 / Ki-67-positive activated islet proliferating cells (AIPCs) for functional insulin production derived from isolated pancreatic islets, and for expanding islet-derived AIPCs in vitro using a culture medium containing an activator. Additionally, the present invention discloses the use of AIPCs for in vivo therapy, particularly for transplantation into mammals for pancreatic diseases including type 1 diabetes.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 689,780 filed June 25, 2018, the full text of which is incorporated herein by reference. Background Technology

[0003] Diabetes is a metabolic disease defined by high blood sugar, but it also causes severe damage to numerous body systems, including nerves, blood vessels, eyes, kidneys, and the heart. According to some studies, it is estimated that more than 400 million people worldwide live with diabetes, and the incidence and prevalence of diabetes are increasing globally, particularly in the United States, China, and India.

[0004] Although multiple variants of diabetes exist, two major types are dominant. In type 1 diabetes, the pancreas produces little to no insulin due to the loss of insulin-producing islet beta cells. These insulin-producing islet cells are present in type 2 diabetes, but the amount and efficacy of the insulin produced are low due to insulin resistance. Therefore, despite variants, diabetes can be characterized as islet cell dysfunction.

[0005] Research on diabetes and islet biology has been severely limited due to the inability to culture and expand islet cells. Currently, islet clusters or cells can only be maintained for approximately three weeks in a deteriorated state, which inherently makes long-term research on islet cells impossible. Furthermore, the inability to culture and propagate these islet cells limits the potential of islet cell replacement therapy, a potential treatment for diabetes.

[0006] Currently, the two current standard techniques for generating beta cells to provide a cell source for new drug development and cell transplantation therapy in diabetes are (i) differentiation of non-beta islet cells, such as induced pluripotent stem (iPC) cells or fibroblasts, through genetic reprogramming and / or differentiation and exposure to multiple maturation factors or differentiation of embryonic stem cells into beta-like cells; and (ii) induction of mature beta cell proliferation by the use of therapeutic molecules. The major drawbacks of current approaches are associated with genetic manipulation, differentiation efficiency, and potentially off-target effects of chemicals used to induce beta cell proliferation. Furthermore, most approaches require long-term culture and significant manipulation steps / times, which increase the risk of microbial contamination and significantly impact production costs, ultimately affecting clinical translation.

[0007] Previously, as this was considered to provide an unprecedented cell source for new drug development and cell transplantation therapy in diabetes, several groups attempted to generate insulin-producing pancreatic beta cells from stem cells in vitro. In 2009, a strategy to induce both human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells similar to adult islet beta cells was disclosed in Cell Research. To generate these iPS cells, somatic cells must undergo episomal reprogramming by applying a number of factors (but not limited to) short hairpin RNAs to OCT4, SOX2, KLF4, and p53; this is a multi-step process that takes several weeks to establish. The differentiated human ES cells at the final stage secreted C-peptide in response to glucose stimulation in a manner close to the response of adult human islets. In addition, the co-expression of C-peptide, PDX1, and NKX6-1 in induced insulin-producing cells was observed for the first time in vitro. To generate these cells, multiple reagents / factors are used, and the multi-stage differentiation protocol takes 2 to 3 weeks to generate the cells.

[0008] In 2014, Pagliuca et al. (Cell, Vol. 159: Issue 2, p. 428-439, October 9, 2014) 10 8Beta cells were generated in vitro from human pluripotent stem cells (hPSCs) using a scalable suspension-based culture system capable of generating more than 10 hPSCs and subsequently differentiated cell types. Their protocol takes 4 to 5 weeks and involves a unique combination of sequential culture steps utilizing factors that influence signaling in numerous pathways, including γ-secritase inhibition, as well as signaling by wnt, activin, hedgehog, EGF, TGFβ, thyroid hormone, and retinoic acid.

[0009] During 2015, a group in California converted human fibroblasts to an endoderm cell fate by using episomal reprogramming factors in combination with specific growth factors and compounds (Reference [Nat. Commun. 2016; 7: 100080]). These cells were further differentiated using compounds that promote differentiation and maturation into functional pancreatic beta-like cells in vitro. However, these cells failed to provide a substitute source of beta cells due to their strict manipulation, which requires encapsulation within a device when the cells are implanted in vivo, the inability to generate all cells in the islets necessary for proper regulation of blood glucose, and the formation of teratomas within 7 weeks after implantation.

[0010] Induction of mature beta-cell proliferation has previously been achieved using small molecule inhibitors of bispecific tyrosine-regulated kinase 1A (DYRK1A) (Literature [Wang, et. al[ , Cell Metab. 2019; 29: 638-65]). The use of DYRK1A inhibitors results in a proliferation index of 1.5% to 3% in beta cells. More recently, to promote the proliferation of mature beta cells, Wang et al. combined pharmacological inhibition of DYRK1A with transforming growth factor beta superfamily (TGFbSF) / SMAD signaling to induce a significant and additional synergistic increase in human beta cell proliferation (average labeling index of 5% to 8% and labeling index as high as 15% to 18%) and an increase in both mouse and human beta cell numbers. To induce the proliferation of mature beta cells, the entire islet is used, and no cells are generated outside the islet. The cells are fixed and remain within the islet structure. To release cells from the islet, the islet was dissociated using 0.05% trypsin neutralized by adding an equal volume of 100% FBS. While this work appears promising, the observed proliferation levels are not sufficient to generate the number of insulin-producing cells required to create a viable source of insulin-producing cells for therapeutic use, such as diabetes treatment.

[0011] Both current standard techniques used to generate genuine beta cells as a source for cell therapy or transplantation in diabetes are intensive, require multiple steps and numerous factors, and take weeks to produce the target cells. Cell-based therapies offer the potential to treat diseases such as Type 1 diabetes and alter the course of these conditions, but this has not been adequately addressed by existing medications. The problem to be solved

[0012] The compositions and methods described herein overcome the problems of the methods described above. The present invention discloses a population of activated islet proliferating cells (AIPCs) and a method for producing the same. The AIPC population can produce insulin and can enable the expansion of the pancreatic islet population to meet the requirements for use in cell-based therapies. means of solving the problem

[0013] The method described herein provides a rapid means for differentiating isolated pancreatic islets into an AIPC population having a specific proportion of islets (as further described herein), said islets that produce insulin and possess both a CD133 cell marker and a Ki-67 marker, which is identified herein as “triple-positive” islets. The so-called “triple-positive” islets are glucose-responsive and secrete both insulin and glucagon in response to appropriate stimulation.

[0014] A representative culture of the above AIPC was deposited as ATCC [Deposit No.: ] in accordance with the Budapest Treaty.

[0015] In addition, the present invention discloses a treatment method comprising the step of administering an effective amount of AIPC population to a mammal, e.g., a human, to recover healthy pancreatic islets capable of producing insulin in response to glucose stimulation. Brief explanation of the drawing

[0016] Figure 1 shows microscopic images of cultured rat islets treated with cell culture medium containing IMG (approximately 3 µg / ml) as further described herein (see Table 2), compared to cultured rat islets cultured in basic medium alone (see Table 3). Within 24 hours of treatment, the treated islets formed fewer clusters, and the islet cells appeared to be actively migrating away from the clusters. The control islets maintained their clustered morphology and showed minimal evidence of cell migration. Around 72 hours of culture, the treated islets lost most of their morphology and began to establish colonies of CD133-positive, Ki-67-positive proliferating islet cells for insulin production (see Example 1). Figure 2 shows microscopic images of cultured human islets treated with cell culture medium containing IMG (approximately 3 μg / ml) compared to untreated control islets. As can be seen from rat islets (shown in Figure 1), cultured human islets treated with cell culture medium containing the activator exhibited the same trend of islet cell migration from the islets (Day 2) and expansion of migrating islet cells into CD133-positive, Ki-67-positive proliferating islet cell colonies for insulin production (Day 7) (see Example 2). Figure 3 shows a bar graph comparing cell viability as determined by FAC after propidium iodide and acridine orange staining for the control cell population (untreated mouse islet cells) versus the treated cell population on days 3 and 10; both populations (control / untreated cells and treated cells) showed cell viability exceeding 80% on days 3 and 10 (see Example 1). Figures 4a and 4b show bar graphs representing the aggregated results of the effects of culture media containing an activator following culture using isolated human islets; culturing human islets in a medium containing IMG (approximately 10 µg / ml) results in an increase in islet cell numbers compared to the control group (untreated human pancreatic islets), as measured by the CyQUANT cell proliferation assay (which measures the DNA content of cells). The assay was performed 3 days after plating the islets, and cell counting via tryptophan blue exclusion was performed on the 10th day after culturing the cells in the medium to measure cell proliferation. Both cell measurements (cell proliferation and cell counting) indicated a significant increase in the number of islet cells derived from human islets cultured in the medium containing the activator; specifically, there were 6,400 cells in the untreated control group compared to 9,500 cells after treatment with IMG (Figure 4a); Compared to approximately 2,500,000 cells after treatment with IMG, there were fewer than 500,000 cells in the untreated control group (Fig. 4b). Figure 5 shows that AIPCs generated by treating isolated non-natural sodium with a culture medium containing an activator (IMG) can expand beyond at least two progeny generations within 5 days. After being labeled with carboxyfluorescein succinimidyl ester (CFSE), the AIPCs were [labeled] with CellTrace TM Analysis was performed 5 days after treatment with a culture medium containing an activator using a CFSE cell proliferation kit. Such kits are used to label cells in vitro and in vivo to track multiple generations using dye dilution via flow cytometry. Within 5 days, two generations of AIPC could be observed. Figures 6a and 6b show that isolated islets cultured in a medium containing the activator (IMG) promote the expansion of islets into populations of cells (AIPCs) containing insulin, CD133, and Ki-67-positive cell phenotypes compared to control islets. Specifically, at least 60% and more than 70% (average) of the isolated islets cultured in the medium containing the activator produced CD133 / Ki-67 / insulin-positive AIPCs (61.47%) (see Figure 6b); whereas control cells showed CD133 / Ki-67 / insulin-positive (essentially 0%) in less than 5% of the control cell population (see Figure 6a). Cultured AIPCs were evaluated for the expression of CD133 (a marker of precursor somatic cells) and Ki-67 (a nucleoprotein associated with proliferation) and intracellular insulin expression using FAC (see Examples 1 and 2). Figure 7 shows that AIPCs derived from human islets isolated after treatment with a culture medium containing an activator secrete insulin following glucose stimulation. A static incubation glucose-stimulated secretion assay was applied to the AIPCs (see Example 2). Insulin secretion from the medium for beta-cell function was evaluated by applying five stimulation conditions to islet-derived AIPCs. AIPCs were subjected to (1) 2.8 mM glucose (control baseline); (2) 16.7 mM glucose (stimulation for insulin); (3) 16.7 mM glucose + 100 μM IBMX (i.e., 3-isobutyl-1-methyl-xanthine; maximal stimulation for insulin); and (4) 1.7 mM glucose + 1 μM epinephrine (stimulation for glucagon); and (5) incubated for 30 minutes with either 5.6 mM glucose + 20 mM KCl (additional stimulation for insulin and glucagon). After the assay, the medium was stored at -20°C and a standard ELISA assay for insulin was performed, and the results showed that insulin secretion was at a level of 11 μIU / mL insulin / million cells for Group 1; at a level of 57 μIU / mL insulin / million cells for Group 2; at a level of 221 μIU / mL insulin / million cells for Group 3; at a level of 6 μIU / mL insulin / million cells for Group 4; and at a level of 22 μIU / mL insulin / million cells for Group 5. Figure 8 shows that AIPCs derived from human islets isolated after treatment with a culture medium containing an activator secrete glucagon following glucose stimulation. A static incubation glucose-stimulated secretion assay was applied to the AIPCs (see Example 2). Glucagon secretion from the medium for alpha cell function was evaluated by applying five stimulation conditions to islet-derived AIPCs. AIPCs were subjected to (1) 2.8 mM glucose (control baseline); (2) 16.7 mM glucose (stimulation for insulin); (3) 16.7 mM glucose + 100 μM IBMX (maximum stimulation for insulin); and (4) 1.7 mM glucose + 1 μM epinephrine (stimulation for glucagon); (5) Incubated for 30 minutes with either 5.6 mM glucose or 20 mM KCl (additional stimulation for insulin and glucagon). After the assay, the medium was stored at -20°C and a standard ELISA assay for glucagon was performed. The results showed that glucagon secretion was 32 pg glucagon / million cell level for Group 1; 30 pg / million cell level for Group 2; 52 pg / million cell level for Group 3; 98 pg / million cell level for Group 4; and 75 pg / million cell level for Group 5. Figure 9 shows bright-field microscopy of islet-derived AIPCs on day 20 of culture, after which islet-derived AIPCs showed signs (visible in microscopic examination and upon staining) that the AIPCs began to generate secondary structures resembling islets. These islet-like structures were measured to be dithizone (DTZ) positive upon testing, indicating putative beta cells in new islets. DTZ binds to zinc ions present in the beta cells of the islets, thereby staining the islets red (color results not shown). Figure 10a shows the FAC marker profile and morphology of islet-derived AIPCs on day 60 after treatment with a culture medium containing the activator (IMG). After 60 days of culture, the profile of the AIPCs was evaluated (using flow cytometry as described in Example 2), and the markers were evaluated as a percentage (%) of cell types. The marker profiles (as a percentage (%) of cells) are as follows: Insulin-positive / Ki-67-positive / CD133-positive cells accounted for 61.47% of the total cell population. Approximately 10% of the cells were negative for the marker profiles. Furthermore, islet-derived AIPCs expanded according to the method of the present invention maintained their marker profiles (CD133 / Ki-67 / insulin "triple" positive phenotype) and cell morphology for a period exceeding 60 days of culture (Figures 10b and 10c). Figure 11 shows that AIPC remains viable in vivo and causes a decrease in blood glucose levels in a hyperglycemic mouse model (see Example 4). Four days prior to AIPC injection, C57BL / 6J mice (n = 6; denoted as M1, M2, M3, M4, M5, and M6) were treated with STZ at a concentration of 75 mg / kg. On day 0, if an animal had a fasting BG level greater than 200 mg / dL, a cell suspension containing approximately 4 million rat AIPCs (isolated from Tomato Red mice) was injected into each STZ-treated hyperglycemic mouse. Fasting blood glucose levels in each STZ-treated mouse were measured twice weekly; two animals were sacrificed on day 28 after AIPC administration, and two animals were sacrificed on day 42. On day 46, an additional (approximately) 10 million cells were injected into the remaining two STZ-treated animals (each via tail vein), and they were sacrificed on day 70. All STZ-treated animals injected with AIPC showed improvement, with blood glucose levels dropping from 300 mg / dL to 500 mg / dL prior to AIPC administration to approximately 200 mg / dL on day 50 after AIPC administration. Figure 12 shows that according to histological analysis of sacrificed animals (Example 4), donor tomato red AIPC is present in the pancreas of STZ-treated animals, suggesting migration / homing of AIPC injected into the tail through the bloodstream flowing to the pancreas. AIPC appears to have higher concentrations over longer periods. Figure 13 shows that according to immunohistological analysis (Example 4) of AIPC-injected mice sacrificed on day 70, regions within the pancreas are marked as "double positive" for both Tomato Red and Ki-67, indicating that AIPC homed within the pancreas can divide and spread. Specific details for implementing the invention

[0017] The present invention describes a composition and a method for promoting the proliferation, expansion, and differentiation of primary cell isolates containing pancreatic islets into an endocrine progenitor cell population containing cells positive for the markers CD133 (a marker for stem cells) and Ki-67 (an active proliferation marker), and for promoting insulin secretion. The composition comprises a culture medium containing a base medium and an activator, wherein the activator comprises, for example, a polypeptide isolated according to SEQ ID NO. 1 or an active fragment thereof. Alternatively, the activator may comprise, for example, a polypeptide isolated according to SEQ ID NOs 2 to 4. The method comprises the use of a culture medium to promote islet cell differentiation and to promote the proliferation of an endocrine progenitor cell population containing marker profiles positive for CD1331 and Ki-67 from isolated primary cells containing pancreatic islets.

[0018] The present invention describes an endocrine progenitor cell population (referred to as AIPC, AIPCs, or AIPC population), wherein the endocrine progenitor cell population is produced by treating isolated primary cells comprising a) of mammalian origin; b) positive for the CD133 cell marker; c) positive for the Ki67 cell marker; d) producing insulin in response to stimulation; and e) pancreatic islets with an activator comprising a polypeptide or polypeptide fragment having amino acid sequence identity according to, for example, SEQ ID NOs 1 to 4.

[0019] Additional methods include the use of endocrine progenitor cell populations generated by the methods described herein for cell transplantation or injection, or for manipulating tissues useful for treating various diseases or conditions of the pancreas.

[0020] The following terms are used in this disclosure to describe different aspects of the invention. These terms are used for illustrative purposes only and are not intended to limit the scope of any aspect of the invention.

[0021] As used herein, "active agent" refers to a protein, polypeptide, peptide fragment, or an analog thereof, which includes any modification thereof and has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to the amino acid sequence according to SEQ ID NO. 01, which includes SEQ ID NO. 2 to SEQ ID NO. 4 representing the fragment of SEQ ID NO. 1. Additionally, a peptide fragment or an analog thereof is also considered, which includes any modification thereof and has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to the amino acid sequence according to SEQ ID NO. 2 to SEQ ID NO. 4.

[0022] As used herein, the terms AIPC, AIPCs, or AIPC population refer to a non-naturally derived endocrine (pancreatic) progenitor cell population generated according to the method described herein, wherein the cell population is positive for the cell markers CD133 and Ki67 and produces insulin in response to stimulation.

[0023] As used herein, the term "growth" refers to the maintenance of cells in a living state and may include, but is not limited to, the proliferation and / or differentiation of cells. The term "propagation" refers to an increase in the number of cells present in the culture medium as a result of cell division.

[0024] As used herein, “culture,” “cultured,” or “culture” refers to removing or isolating cells from an environment (such as a host mammal) and growing them in a desirable artificial environment within a test tube. “Cultured cells” is intended to include sub-culturing (i.e., passage) by transferring cells to a new container containing fresh growth medium to provide additional space for continued growth, differentiation, and / or proliferation.

[0025] The term "expanded" is intended to mean that the obtained cell population originated from the ex vivo culture medium of pancreatic islets in a medium composition containing IMG. The term "expanded" is not understood or limited by any mechanism or theory regarding cell origin and may include cells of new origin at the time of culture.

[0026] The reference to “pancreatic cells” includes these cells normally found in the pancreas, and includes pancreatic islet cells, e.g., alpha cells for glucagon synthesis, beta cells for insulin production, and any combination thereof. Islet-derived AIPCs diffused from pancreatic islet cells cultured by the method described herein are useful for producing insulin, among other things, and for injecting into an individual to treat diabetes (e.g., type 1 diabetes). The pancreatic cell population generally has 3% or less positive for Ki-67, a cell marker used to distinguish AIPCs generated from pancreatic cells using the method described herein.

[0027] As used herein, the term “target site” refers to an area within a recipient host (mammal, preferably human) where treatment or replacement is required. The target site may be a single area within a specific organ or multiple areas within the host. In some embodiments, replacement or replacement results in the same physiological response as normal tissue, such as pancreatic tissue, whether or not the pancreas is targeted.

[0028] As used herein, “treating,” “to treat,” or “treatment,” and other grammatical equivalents as used herein, include alleviating, weakening, or improving symptoms of a disease or pathology, preventing additional symptoms, improving or preventing the metabolic cause underlying the symptoms, suppressing the disease or pathology, e.g., preventing the onset of the disease or pathology, alleviating the disease or pathology, causing the regression of the disease or pathology, alleviating the pathology caused by the disease or pathology, or stopping the symptoms of the disease or pathology, and include prevention. These terms further include achieving therapeutic benefits and / or preventive benefits. Therapeutic benefits mean the eradication or improvement of the underlying disease to be treated. Additionally, therapeutic benefits are achieved by the eradication or improvement of one or more physiological symptoms associated with the underlying disease such that improvement is observed in the patient, even if the patient is still suffering from the underlying disease.

[0029] As used herein, “therapeutic effective dose” or “effective dose” refers to an amount sufficient to achieve the stated effect. A therapeutic effective dose for treating a condition is an amount of active substance capable of achieving a clinically relevant endpoint in a patient or patient population. As a non-limiting example, an effective dose composition containing AIPC is administered in an amount of about 4 million to about 14 million cells / kilogram or more than 200 million cells to lower a hyperglycemic individual (e.g., a patient suffering from hyperglycemia) to less than 150 mg / dL to a normal blood glucose level, i.e., approximately 100 mg / dL to 125 mg / dL (5.6 to 6.9 mmol / L). The appropriate dosage of AIPC formulated as a composition for injection or infusion will depend on the individual to be treated and the severity of the condition to be treated. The embodiments disclosed herein were performed in part in mouse models, and cell culture experiments were performed using mammalian (human or mouse) cells. By using a scaling method such as allometric scaling, it is possible to predict an appropriate and exemplary dosage range for administering a composition containing AIPC as disclosed herein to adult humans. Dose scaling is an empirical approach and is well characterized and fully understood in the art. According to this approach, some unique features regarding anatomical, physiological, and biochemical processes exist between species, and it is presumed that possible differences in pharmacokinetics and physiological time are explained by scaling. As one example not intended to be limiting, based on the literature, the human pancreas has between 600,000 and up to 2 million islets; consequently, a normal human pancreas contains approximately 600 million islet cells, half of which are beta cells.Therefore, based on these metrics, a dosage of 4 million AIPCs / kg can be expected to be sufficient to replace beta cells in a normal human pancreas.

[0030] As used herein, the term "sequence identity" refers to the identity between two or more amino acid sequences expressed in terms of identity or similarity between sequences. Sequence identity can be measured in terms of the identity ratio (%); the higher the ratio (%), the more identical the sequences are. The identity ratio (%) is calculated over the total length of the sequence. Homologs or orthologs of amino acid sequences have a relatively high degree of sequence identity when aligned using standard methods. This homology is due to the ortholog proteins being from more distantly related species (e.g., humans and Chamaedorea elegans ( C. elegans It is more significant when derived from species that are more closely related (e.g., human and mouse sequences) compared to the ) sequence.

[0031] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms [Smith & Waterman, Adv. Appl. Math. 2: 482, 1981]; [Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970]; [Pearson & Lipman, Proc. Nat. Acad Sci. USA 85: 2444, 1988]; [Higgins & Sharp, Gene, 73: 23744, 1988]; [Higgins & Sharp, CABIOS 5: 151-3, 1989]; [Corpet et al. , Nuc. Acids Res. 16: 10881-90, 1988]; Literature[Huang et al.Computer Appls. in the Biosciences 8, 155-65, 1992]; and literature [Pearson et al. It is described in the literature [Altschul et al. Detailed considerations regarding sequence alignment methods and homology calculations are presented in [J. Mol. Biol. 215:403-10, 1990]. The level of sequence identity can be measured using NCBI's Basic Local Alignment Search Tool (BLAST) (Literature [Altschul et al. [ , J. Mol. Biol. 215: 403-10, 1990]), this is available from several sources including the National Center for Biotechnology Information (NCBI; National Library of Medicine, Room 38A 8N805, Building 38A, Methussdale, Maryland, USA) and is available on the Internet.

[0032] The activator may be a polypeptide comprising a peptide or a peptide fragment thereof consisting of 293 amino acids, for example, a peptide fragment as described in U.S. Patent Application No. 15 / 811,060, or a combination thereof; wherein the sequence of the polypeptide or fragment has at least 50% homology with a portion of the (293) amino acid sequence shown below and represented by SEQ ID NOs. 1 to 4:

[0033] [Table 1]

[0034] Sequence Nos. 1 to 4:

[0035] Sequence No. 01 (293 amino acids; fragment of approximately 31 kDa)

[0036] MADDAGAAGGPGGPGGPGMGNRGGFRGGGFGSGIRGRGRGRGRGRGRGRGRGRGG

[0037] KAEDKEWMPVTKLGRLVKDMKIKSLEEIYLFSLPIKESEIIDFFLGASLKDEVLKIMPVQK

[0038] QTRAGQRTRFKAFVAIGDYNGHVGLGVKCSKEVATAIRGAIILAKLSIVPVRRGYWGNK

[0039] IGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTAT

[0040] LGNFAKATFDAISKTYSYLTPDLWKETVFTKSPYQEFTDHLVKTHTRVSVQRTQAPAVA

[0041] TT

[0042] Sequence No. 2 (159 amino acids; fragment of approximately 17 kDa)

[0043] GHVGLGVKCSKEVATAIRGAIILAKL S IVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVR

[0044] LIPAPRTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGNFAKATFDAISKTYSYLTPD

[0045] LWKETVFTKSPYQEFTDHLVKTHTRVSVQRTQAPAVATT

[0046] Sequence No. 3 (75 amino acids; fragment of approximately 8 kDa)

[0047] SIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGI

[0048] DDCYTSARGCTATLGN

[0049] Sequence No. 4 (87 amino acids; fragment of approximately 9 kDa)

[0050] GHVGLGVKCSKEVATARGAIILAKLSIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLV

[0051] RLIPAPRGTGIVSAPVPKKLLMMAGIDD

[0052] Amino acid residues of the activator may be modified post-translationally or conjugated with other functional or non-functional molecular groups. For example, literature describing the antagonistic citrullination and methylation of human ribosomal protein S2 (e.g., SEQ No. 1) [Guo et al. See Mol. Biosyst. 7(7): 2286-2295, 2011. Naturally, such modified amino acid residues are included in amino acid sequences and are within the range of active agents described herein.

[0053] For example, polypeptides and / or polypeptide fragments according to sequence numbers 1 to 4 may be produced under conditions known in the art for protein production, such as production in bacteria, yeast, or by synthetic means, or as described in U.S. Patent Application No. 15 / 811,06.

[0054] A first embodiment relates to a composition useful for stimulating the growth, diffusion, and differentiation of pancreatic islet populations into AIPC populations, said composition comprising a culture medium comprising a base medium and an effective amount of an activator, said activator comprising a polypeptide or an active fragment thereof comprising an amino acid sequence according to one or more of SEQ ID NOs 1 to 4 (listed in Table 1). In one embodiment, the polypeptide comprises an amino acid sequence having at least 50% sequence identity with respect to the amino acid sequence disclosed in SEQ ID NO. 01; in another embodiment, the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to the amino acid sequence disclosed in SEQ ID NO. 1. Alternatively, the polypeptide comprises an amino acid sequence having at least 50% sequence identity with respect to any one of SEQ ID NOs 2 to 4; In another embodiment, the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to the amino acid sequence disclosed in any one of SEQ ID NOs 2 to 4.

[0055] A second embodiment relates to a method for generating an AIPC population, said method comprising the step of culturing a pancreatic islet population in a test tube with a cell culture medium containing a basic medium and an effective amount of an activator. The composition may be analyzed to measure the responsiveness of the composition to glucose, such as the ability to secrete glucose when exposed to a specific amount of glucose. The characteristic of the AIPC population is the ability to secrete glucagon when exposed to an appropriate stimulus.

[0056] A third embodiment relates to a composition comprising an activated islet proliferating cell (AIPC) population, wherein 60% of the AIPC population consists of CD133 cell markers and Ki-67 cell markers, and at least 60% of the AIPC population is capable of producing insulin.

[0057] A fifth aspect relates to a composition comprising a population of activated islet proliferating cells (AIPC) obtained by a method comprising the step of culturing a pancreatic islet population in vitro with a cell culture medium containing a basic medium and an effective amount of activator.

[0058] A sixth aspect relates to a method for generating an AIPC population, the method comprising the steps of: obtaining an AIPC population by culturing a pancreatic islet population in vitro with a cell culture medium containing a basic medium and an effective amount of activator; screening the AIPC population for one or more cell markers selective for CD133, Ki-67, and insulin; and collecting an AIPC population confirmed by screening to be CD133 / Ki-67-positive and insulin-producing from the cultured cell population. The method for generating an AIPC population may further comprise the steps of plating the AIPC population onto a suitable medium; obtaining a cultured AIPC population by treating the cells with a culture medium containing a basic medium and an effective amount of activator; and subculturing the cultured AIPC until the cultured AIPC population of CD133 / Ki-67-positive and insulin-producing cells spreads to a suitable percentage (%). Additionally, CD133+ / Ki-67+ / insulin+ ("triple-positive") islet cells can be packaged, formulated, or encapsulated for delivery or transplantation into mammals (e.g., humans).

[0059] The seventh aspect relates to a method for expanding and spreading an activated islet proliferating cell ("AIPC") population, the method comprising the steps of enzymatically desorbing a cultured AIPC population to obtain a desorbed AIPC population, and plating a composition comprising the desorbed AIPC population and a cell culture medium comprising a base medium and an activator onto a culture plate. A suitable protease, e.g. trypsin, may be used for the expansion and spreading of the AIPC. Once desorbed, the AIPC may be centrifuged for a sufficient period (e.g., 7 minutes) at a rotation speed (e.g., 1000 rpm) sufficient to form a pellet. It may be preferable to plating the desorbed AIPC at a suitable cell density, e.g., about 1,000 cells / cm². An interesting feature of the AIPC, including the AIPC population, the desorbed AIPC, etc., is viability for at least 100 days after isolation.

[0060] The eighth aspect relates to a population of activated islet proliferating cells (“AIPC”) in the form of a packaged or encapsulated formulation for administration or infusion into a mammal for in vivo therapy, specifically for the treatment of one or more pancreatic diseases, specifically type 1 diabetes. The AIPC population may be packaged as a delivery solution or packaged within a delivery vehicle and may be administered by implantation, injection, or infusion, whether administration is systemic or local or induced at a target site.

[0061] A ninth embodiment relates to a method for treating pancreatic disease in mammals, wherein the pancreatic disease is hyperglycemia or type 1 diabetes mellitus, and the method comprises the step of obtaining an AIPC population by culturing a population of pancreatic islets from a mammalian species in vitro in a culture medium containing a basic medium and an effective amount of activator. In one embodiment, the AIPC population consists of CD133 / Ki-67-positive cells for insulin production. In another embodiment, the method for treating pancreatic disease further comprises the step of measuring the response of the AIPC population to glucose. In yet another embodiment, the method for treating pancreatic disease further comprises the step of expanding and spreading the AIPC population; and further comprises the step of transplanting a composition comprising the AIPC population (e.g., a population of mostly CD133 / Ki-67-positive cells for insulin production) into a mammal, and the step of delivering the composition to a target site to provide treatment for pancreatic disease. In one embodiment, the composition may be delivered as an aqueous solution, suspension, encapsulation, microencapsulation and / or encapsulation or semi-solid formulation, wherein the composition may be delivered to a mammal via one or more of injection, infusion, omental pouch or peritoneal pouch, or surgical implantation, or delivered to a target site within the mammal by packaging the composition as part of a device.

[0062] In another aspect of the present invention, the composition comprises a population of activated islet proliferating cells (AIPC) and further comprises one or more buffers, one or more pharmaceutically acceptable carriers, or one or more pharmaceutically acceptable additives.

[0063] The cell culture system described herein comprises, at least initially, a pancreatic islet population; a cell culture growth substrate; and a culture medium comprising a basic medium and an effective amount of an activator, wherein the activator comprises, for example, a polypeptide according to SEQ ID NOs 1 to 4 or a fragment thereof, and the polypeptide or polypeptide fragment has at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity with the polypeptide according to SEQ ID NO. 1, for example, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0064] Examples

[0065] The following examples are provided as examples but are not intended to limit the subject matter claimed herein. Throughout the examples, the term “IMG” is used to refer to a specific activator identified herein as SEQ ID NO. 1, provided that other activators, e.g. SEQ ID NOs 2 through 4, may be used as described and defined herein. The use of the term IMG and the amount thereof do not imply any specific dosage or concentration of the activator. In the examples, the concentrations of the additives / supplements and activators in the culture medium are described in Table 2, while the control medium is listed in Table 3.

[0066] Culture conditions

[0067] The cell culture medium implemented under the example was incubated at 37°C under standard CO2 (5%) conditions, and culture (cell plating and division) was performed in a vertical laminar flow hood under standard aseptic techniques and conditions or using standard aseptic techniques and conditions. Unless otherwise noted, AIPCs were cultured in the media described in the table. AIPCs were divided when they reached approximately 70% to 80% confluency during culture.

[0068] The splitting technique involved removing the supernatant from the culture plate (the supernatant was preserved). Subsequently, the plate was washed with 2 ml to 5 ml of PBS (the wash solution was preserved). AIPC was desorbed using approximately 3 ml to 5 ml of trypsin (e.g., available from Sigma-Aldrich) by incubating the cells in the presence of trypsin at 37°C for approximately 3 to 5 minutes until the cells desorbed. Subsequently, the plate was washed again with PBS. Then, the trypsin-treated cells, the preserved PBS wash solution, and the collected cell culture supernatant were centrifuged at 1,000 rpm for 7 minutes at 4°C. The obtained supernatant was decanted, the pellet was resuspended in 2 ml of PBS, and centrifuged again. Next, the supernatant was removed, the pellet was resuspended in a culture medium containing IMG, and plated again at a cell density of ~1,000 cells / cm².

[0069] culture medium Media components sheep CMRL badge 500 ㎖ L-glutamine 2 mmol Ciproproxacin amphotericin B penicillin stretto 2 mg / ℓ 0.1 mg / ℓ 100,000 units / ℓ 100,000 micrograms / ℓ Polypeptide of 293 amino acids (IMG) according to sequence number 01 3 µg / ml to 20 µg / ml bovine fetal serum (FCS) 10%

[0070] Control group media Media components sheep CMRL badge 500 ㎖ Ciprofloxacin 2 mg / ℓ Amphotericin B 0.1 mg / ℓ penicillin 100,000 units / ℓ Streptomycin 100,000 micrograms / ℓ bovine fetal serum (FCS) 10%

[0071] Example 1

[0072] In one exemplary study, 300 rat islets were isolated from the pancreases of 8 to 10-week-old male BALB / c mice (e.g., available from Jackson Labs or Charles River). The isolated islets were cultured in culture medium containing CMRL medium supplemented with 10% fetal bovine serum, 2 mmol L-glutamine, antibiotics, and IMG at a concentration of about 3.0 µg / ml (originally developed by Connaught Medical Research Laboratories, and now available from Mediatech). The treated cells were grown together and compared to control cells, which were the same cells grown in a basic medium without IMG. Within 24 hours, based on visual inspection by microscopy at 10x magnification (using a Leica Light Microscope equipped with a Lumenera camera featuring Infinity Analyze software), cells grown in cell culture medium containing IMG were observed to form fewer clusters, and the cultured islet cells appeared to be actively migrating away from the cell clusters. In contrast, control islet cells were observed to maintain their clustered form and show minimal evidence of cell migration. Three days after treatment, islet cells cultured in medium containing IMG exhibited both free-floating and attached cells within the culture medium. 30% of the free-floating cells were viable, as measured by acridin orange (AO) and propidium iodide (PI) staining. AO is an intercalating dye that can penetrate both living and dead cells, whereas PI is a cell-impermeable fluorescent solution used to exclude non-viable cells in flow cytometry.PI binds to double-stranded DNA via insertion between base pairs but is excluded from cells with intact plasma membranes. 85% of the attached cells were viable. In contrast, the control islet cell culture showed no signs of free-floating or attached cells, even though the islet clusters appeared healthy, as measured by cell viability exceeding 90%. By around day 10, islet cells cultured in medium containing IMG exhibited a low percentage of islet cell clusters; free-floating cells were not distinguishably present during culture, and attached cells appeared to form colonies. The total cell viability of islet cells cultured in medium containing IMG was measured to be over 90%. Even though the control cells continued not to show migration or attachment, the cell viability of the islet clusters remained at approximately 90%. On day 25 of culture, islet cells from both the treated and control (untreated) cultures were fixed and stained for CD133 (a marker for precursor somatic cells) and insulin. Immunohistochemical staining revealed that at least 75% of islet cells treated with cell culture medium containing IMG were insulin-positive. Additionally, at least 70% of the treated islet cells were CD133-positive; and approximately 60% or more of the treated islet cells were tested as positive for both CD133 and insulin (considered "double-positive"). In contrast, 90% of the control cells were insulin-positive, while less than 5% of the cell population was positive only for the marker CD133. Similarly, less than 5% of the cell population was found to be double-positive for both the CD133 marker and insulin.

[0073] According to the results, the cell culture system and medium described herein were found to have the effect of inducing the activation and migration of islet cells out of the islets. These cells form colonies of CD133 / insulin "double-positive" cell populations presumed to exhibit activated beta cell precursors, which can subsequently be used in methods and therapies for regenerating damaged islets.

[0074] Example 2

[0075] In another exemplary study, human snot was used. Human snot for research (HIR), which is a primary human snot processed from an organ donor pancreas. ®Human islets such as those shown are commercially available from Prodo Laboratories, Incorporated. Generally, less than 3% of human islet populations are positive for Ki-67, and approximately 50% of healthy islets produce insulin in response to stimulation. In this example, 10 islets were cultured in a culture medium containing CMRL 1066 medium, 10% fetal bovine serum, 10% human serum, 2 mmol / L-glutamine, antibiotics, and IMG at a concentration of about 10.0 μg / mL. Islets cultured in the culture medium containing IMG were compared to control cells cultured in CMRL 1066 medium without IMG (alone). After culturing the treated and untreated human islets at 37°C for 5 days, these islets were subjected to the CyQUANT cell proliferation assay (e.g., available from Invitrogen). Within 5 days, human islets cultured in a culture medium containing IMG were found to have approximately 1.5 times the cell count compared to the control group (9,469 cells / mL in islets cultured with IMG compared to 6,363 cells / mL in the control group) (p < 0.001). Human islets cultured in a medium containing IMG exhibited the same trend observed in mouse islets through microscopic examination (Example 1). Specifically, it was found that the smaller the islet size, the less tissue structure there was, and as a result, islet cells appeared to actively migrate away from the clusters.

[0076] Example 3

[0077] Separately, human sores (3,000 to 10,000) were cultured in control medium or CMRL 1066 medium (supplemented with 10% fetal bovine serum, 10% human serum, 2 mmol / L glutamine, antibiotics, and 10.0 μg / mL IMG) on plates coated with an adhesion factor mixture (AFM) containing type 1 collagen (collagen from rat tails; Sigma-Aldrich C3867) and endothelial cell adhesion factor (ECAF; Sigma-Aldrich E9765). Various ratios of ECAF and collagen, including a 50 / 50 ratio of collagen to ECAF, may be used. An AFM thin layer (between 3 mL and 10 mL) was applied and set for 30 minutes, after which excess AFM was removed, and the plates were left to dry in a hood for 45 minutes. Before use, the plate was washed with PBS to remove any potential contaminants. Sodium cultured in an AFM-treated flask (plates may be used) was incubated in either culture medium or control medium for 2 to 5 days.

[0078] The control islets (cultured in standard CMRL medium without IMG) were shown to maintain their cluster morphology based on results observed using bright-field microscopy and showed no evidence of cell migration. Intracellular insulin expression, as well as the expression of CD133 and Ki-67 (nuclear proteins associated with proliferation), was evaluated in islet cultures (whether cultured in medium containing IMG or in standard medium) by fluorescence-activated cell sorting (FACS) using a BD FACSAria flow cytometry instrument. First, cultured cells were labeled for CD133 expression, then fixed according to the manufacturer's instructions, permeated with FOXP3 fixation / permeation buffer, and stained with fluorescence assay antibodies conjugated for Ki-67 and intracellular insulin, respectively. Subsequently, FACS analysis was performed after FOXP3 fixation / permeation and staining with conjugated fluorescence assay antibodies. Human islet cells cultured in a medium containing IMG were found to be positive (triple positive) for CD133, Ki-67, and insulin, specifically more than 80% positive for insulin, CD133, and Ki-67, whereas control cells were found to be more than 75% positive for insulin and approximately 10% to 15% positive for CD133 and Ki-67.

[0079] To test the glucose responsiveness of cultured islets, a static incubation glucose-stimulated secretion assay was applied to AIPCs. Approximately 1 x 10⁶ per well was used in a 6-well dish. 6Cells were plated, and these cells were subjected to five stimulation conditions to evaluate insulin secretion from the medium for beta-cell function and glucagon secretion for alpha-cell function. Cells were incubated for 30 minutes with one of the following: (1) a buffered solution of KREB supplemented with glucose at a physiological concentration of 2.8 mM (control baseline); (2) 16.7 mM glucose (stimulation for insulin); (3) 16.7 mM glucose + 100 μM IBMX (maximum stimulation for insulin); (4) 1.7 mM glucose + 1 μM epinephrine (stimulation for glucagon); and (5) 5.6 mM glucose + 20 mM KCl (additional stimulation for insulin and glucagon). After the assay, the medium was stored at -20°C and subjected to standard ELISA assays for insulin and glucagon. As shown in Figures 7 and 8, islets cultured in a cell culture medium containing IMG were found to secrete insulin in response to glucose stimulation.

[0080] Example 4

[0081] To determine whether AIPC is viable in vivo and to demonstrate AIPC as a treatment for hyperglycemia and a method for blood glucose control, six C57BL / 6J mice (Jackson Lab; stock number: 000664) were treated with streptozotocin (STZ) (75 mg / kg), a chemical used to destroy insulin-producing cells and induce a type 1 diabetes phenotype in mice. Once STZ-treated animals (n = 6) exhibited fasting blood glucose levels exceeding 200 mg / dL, an aqueous suspension of phosphate-buffered saline (PBS) or a cell suspension containing approximately 4 million AIPCs in 300 µl of PBS was administered to the STZ-treated mice by tail vein injection (4 days after treatment with STZ). The AIPC used in this study was isolated from tomato red mice (Jackson Lab; stock number: 007576) in which cells express strong td-tomato fluorescence.

[0082] To generate AIPC for cell suspension, the previously disclosed method (literature [Bertera et. alMouse sacs were isolated from Tomato Red mice by type 5 collagenase digestion (available from Sigma-Aldrich) in accordance with [J Transplant. 2012; 2012: 856386, Dec 9, 2012]. Briefly, immediately after sacrifice, 2 to 3 ml of cold collagenase solution (1.95 mg / ml in Hank's Balanced Salt Solution (HBSS)) was injected into the pancreas via the common bile duct. The fully dilated pancreas was removed, and the tissue was incubated in a tissue culture flask at 37°C for 20 minutes, followed by shaking for 5 seconds to break the tissue. The digested tissue was washed with cold HBSS supplemented with 0.2% BSA, and the sacs were purified using a Ficoll gradient. Selected sacs were cultured in culture medium (Table 2) at 37°C under standard CO2 (5%) conditions. T-75 tissue culture plates were pretreated with AFM (ECAF and type 1 collagen) before use as disclosed herein. Approximately 300 to 500 islets per T-75 were plated onto the culture medium (Table 2). As early as 24 hours after plated, cells began to migrate out and the islets began to attach to the plate; by about 72 hours, cells had migrated out and most of the islets had attached; and on approximately day 7 to day 10, the AIPCs had confluenced. On day 14, the AIPCs underwent continuous subculture. Subsequently, the cultured IPCs were collected for injection into STZ-treated mice.

[0083] Fasting blood glucose levels in STZ-treated mice were measured twice weekly following the administration of AIPC; two animals (M2 and M6) were sacrificed on day 28, and two animals (M3 and M5) were sacrificed on day 42 after AIPC injection. On day 46, an additional 10 million cells per 300 µL of suspension (PBS) were administered to the remaining two animals (M1 and M4) via tail vein injection, and they were sacrificed on day 70 of the study.

[0084] According to the results, STZ-treated / AIPC-injected mice showed a decrease in fasting blood glucose levels over time. The last two animals (these STZ-treated mice, which received two AIPC injections (approximately 14 million cells total)) showed normal blood glucose levels (blood glucose levels of less than 200 mg / dL) around day 53 (see Fig. 11). In addition, histological and immunological analyses, as indicated by histological staining and microscopic analysis (results shown in Figs. 12 and 13), proved that AIPC injected into STZ-treated mice via the tail vein homed to the pancreas and engrafted within it, and continued to spread once engrafted.

[0085] Histological analysis of sacrificed animals revealed the presence of donor Tomato Red AIPC in the pancreas of STZ-treated animals, suggesting that AIPC injected via tail vein migrated / homed to the pancreas (see Fig. 12). The concentration of AIPC appears to increase with longer duration. Immunohistological analysis of STZ-treated mice sacrificed on day 70 after receiving AIPC via tail vein injection showed that regions within the pancreatic tissue were double-positive for both Tomato Red and Ki-67 (based on histological staining for markers), indicating that the injected AIPC homed to the pancreas, where continuous cell division and proliferation were possible (see Fig. 13).

[0086] The method described herein comprises the step of culturing isolated islets in a cell culture medium containing a basic medium and an activator. The culture medium showed no evidence of causing a decrease in islet cell viability and showed direct evidence of promoting islet dissociation and migration of islet cells from the inner core of the islets. Consequently, the expansion of these cultured islet cells obtained appeared within a few days when treated with the culture medium. The cells obtained by expanding in the cell culture medium containing the activator tested positive for insulin expression, CD133, and Ki-67, representing a novel AIPC cell population presumed to be generated using the method and culture medium described herein. AIPCs generated by culturing isolated islets in the culture medium containing the activator were serially subcultured for more than 18 generations and were found to remain viable for at least 100 days after isolation.

[0087] A population of activated islet-derived proliferating cells produced according to the method of the present invention comprises at least 30% CD133+ / Ki-67+ cells in culture; or at least 35% CD133+ / Ki-67+ cells in culture; or at least 40% CD133+ / Ki-67+ cells in culture; or at least 45% CD133+ / Ki-67+ cells in culture; or at least 50% CD133+ / Ki-67+ cells in culture; or at least 60% CD133+ / Ki-67+ cells in culture; or at least 70% CD133+ / Ki-67+ cells in culture; or at least 80% CD133+ / Ki-67+ cells in culture; or at least 85% CD133+ / Ki-67+ cells in culture; Or, containing about 90% CD133+ / Ki-67+ cells in culture; at least 60% of the activated islet proliferative cells derived from islets also produce insulin.

[0088] The encapsulation of islet cells generated in vitro and their transplantation into mammals have been previously characterized in the art (e.g., the literature cited herein by reference [Altman, et al. [See , 1984, Trans. Am. Soc. Art. Organs 30: 382-386] and U.S. Patent No. 6,703,017 B1), and may be suitable for AIPC produced according to the method disclosed herein. Preferably, the encapsulating agent is hypoallergenic, is easily and stably positioned within the target tissue, and provides additional protection to the transplanted cell composition, thereby protecting the transplanted IPPC and preventing its destruction.

[0089] Although the invention described above has been described in some detail as examples and embodiments for clarity of understanding, it will be obvious that specific modifications and changes may be made within the scope of the appended claims. It will be obvious to those skilled in the art that features described in connection with any of the above-described aspects and various embodiments may be interchangeably applicable between different embodiments.

[0090] The aspects and embodiments of the present invention described above are examples for illustrating various features of the present invention. All disclosures and patent applications mentioned in this application represent the level of a person skilled in the art to which the present invention pertains. All disclosures and patent applications are incorporated herein by reference to the same extent as each individual disclosure or patent application is incorporated herein by reference in a specific and individual manner.

[0091] Throughout the description and claims of this specification, "comprising" and "containing" and variations thereof mean "comprising but not limited to," and are not intended to exclude (or not exclude) other moiety, additive, component, or step. Throughout the description and claims of this specification, the singular includes the plural unless the context otherwise requires. In particular, where an indefinite article is used, the specification should be understood to consider the plural as well as the singular unless the context otherwise requires.

[0092] Features, features, compounds, chemical moieties, or groups described together with specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, unless they are incompatible with one another. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process disclosed thus disclosed may be combined in any combination, except where at least some of such features and / or steps are mutually exclusive combinations. The present invention is not limited to the details of any of the embodiments described above. The present invention extends to any novel features or any novel combinations among the features disclosed in this specification (including any appended claims, abstract, and drawings), or to any novel steps or any novel combinations among the steps of any method or process disclosed thus disclosed.

[0093] Information disclosed herein, including, for example, U.S. Patent Application No. 15 / 811,060 filed November 13, 2017; PCT / GB2019 / 050049 filed January 9, 2019; and U.S. Provisional Patent Application No. 62 / 689,780 filed June 25, 2018, is incorporated herein by reference. In the event of any conflict between terms and / or expressions cited herein and terms and / or expressions disclosed herein, the information disclosed herein shall prevail.

Claims

Claim 1 A method for generating an activated islet proliferating cell (AIPC) population, comprising the step of culturing a pancreatic islet population in vitro using a cell culture medium containing a basic medium and a polypeptide according to SEQ ID NO. 1 or SEQ ID NO. 2, wherein at least 60% of the AIPC population is positive for the CD133 cell marker and the Ki-67 cell marker; and at least 60% of the AIPC population produces insulin. Claim 2 A method according to claim 1, wherein the pancreatic islet population comprises 10 to 10,000 islets. Claim 3 A method according to claim 1 or 2, wherein the culture takes place over a period of 2 to 10 days. Claim 4 A method according to claim 1 in which at least 80% of the AIPC population is positive for insulin, CD133 and Ki-67. Claim 5 delete Claim 6 The method of claim 1, wherein the cell culture medium comprises a polypeptide according to SEQ ID NO. 1 or SEQ ID NO. 2 at a concentration of 1 μg / ml to 20 μg / ml. Claim 7 The method of claim 1, wherein the cell culture medium comprises a polypeptide according to SEQ ID NO. 1 or SEQ ID NO. 2 at a concentration of 5 μg / ml to 15 μg / ml. Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete

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