Compositions and methods for proliferating insulin-producing islet cells and therapeutic uses thereof

Abstract

Compositions and methods for proliferating insulin-producing islet cells and therapeutic uses thereof are provided. Compositions and methods for differentiating pancreatic cells into functional insulin producing CD133 / Ki-67-positively activated pancreatic islet proliferative cells (AIPCs) derived from isolated pancreatic islets, and amplifying the derived AIPCs in in vitro culture using a culture medium comprising an active agent are disclosed. Also disclosed is the use of AIPC for in vivo therapy, in particular for implantation in mammals for pancreatic conditions, including type I diabetes.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980055757.1, filed on June 20, 2019, entitled “Compositions and methods for proliferating insulin-producing pancreatic islet cells and therapeutic uses thereof”.

[0002] Citation of Related Applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 689,780, filed on June 25, 2018, which is incorporated herein by reference in its entirety. Background Art

[0004] Diabetes is a metabolic disease defined by high blood sugar levels, but it also causes severe damage to multiple body systems, including nerves, blood vessels, eyes, kidneys, heart, etc. Some studies estimate that there may be more than 400 million people living with diabetes worldwide, and the incidence and prevalence of diabetes is increasing globally, especially in the United States, China, and India.

[0005] There are many variants of diabetes, but two main types account for the majority. In type 1 diabetes, the pancreas produces little or no insulin due to the loss of insulin-producing pancreatic β-cells. These insulin-producing islet cells are present in type 2 diabetes, but the amount and effectiveness of the insulin produced are lower due to insulin resistance. Therefore, regardless of the variant, diabetes can be characterized by dysfunction of the islet cells.

[0006] The study of diabetes and pancreatic islet biology has been severely limited by the inability to culture and amplify islet cells. Currently, islet clusters or cells can only be maintained in culture for about 3 weeks, which is essentially in a deteriorating state, thereby hindering long-term research on islet cells. This inability to culture and proliferate islet cells also limits the ability of islet cell replacement therapy as a potential cure for diabetes.

[0007] Currently, two standard technologies currently used to generate β-cells to provide a cell source for drug discovery and cell transplantation therapy in diabetes are: (i) non-β islet cells, such as induced-pluripotent stem (iPC) cells, or fibroblasts through genetic reprogramming and / or exposure to differentiation and multiple maturation factors, or through differentiation of embryonic stem cells into β-like cells; and (ii) induction of proliferation of mature β-cells by using therapeutic molecules. The main drawbacks of current methods are related to genetic manipulation, differentiation efficiency, and potential off-target effects of chemicals used to trigger β-cell proliferation. In addition, most methods require long-term cultivation and significant operating steps / time, which increases the risk of microbial contamination, significantly affects production costs, and ultimately affects clinical translation.

[0008] Previously, the research group has attempted to produce insulin-producing pancreatic β-cells from stem cells in vitro, because it is believed that this will provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. In 2009, a strategy for directing both human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells similar to adult pancreatic β-cells was published in Cell Research. In order to produce these iPS cells, somatic cells must undergo episomal reprogramming by undergoing multiple factors, including (and not limited to) OCT4, SOX2, KLF4 and short hairpin RNA against p53, and this is a multi-step event that takes several weeks to establish. In the final stage, differentiated human ES cells respond to glucose stimulation and secrete C-peptide in a manner close to that of adult pancreatic islets. In addition, the co-expression of C-peptide, PDX1 and NKX6-1 in induced insulin-producing cells was observed in vitro for the first time. In order to produce these cells, a variety of reagents / factors were used, and the procedure required a multi-step differentiation procedure of 2-3 weeks to produce cells.

[0009] In 2014, Pagliuca et al. (Cell, Vol. 159:2, pp. 428-439, October 9, 2014) generated β-cells in vitro from human pluripotent stem cells (hPSCs) using a scalable suspension-based culture system that could generate more than 10 8 Their protocol requires 4-5 weeks and involves a unique combination of sequential culture steps using factors that affect signaling in multiple pathways, including signaling through wnt, activin, hedgehog, EGF, TGFβ, thyroid hormone, and retinoic acid, as well as γ-secretase inhibition.

[0010] And in 2015, a group in California converted human fibroblasts to an endoderm cell fate by using episomal reprogramming factors combined with specific growth factors and compounds (Nat. Commun. 2016;7:100080). These cells were further differentiated using compounds that promoted in vitro differentiation and maturation into functional pancreatic β-like cells. However, these cells have yet to provide a replacement source of β-cells, primarily because of their rigorous manipulation requiring encapsulation of the cells in a device when implanted in vivo, their inability to generate all the cells in the islets necessary for proper blood glucose regulation, and the formation of teratomas within 7 weeks after transplantation.

[0011] Previously, the induction of proliferation of mature β-cells was accompanied by the use of small molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) (Wang et al., Cell Metab. 2019; 29: 638-65). The use of DYRK1A inhibitors resulted in a proliferation index of 1.5%-3% in β-cells. Recently, in order to promote the proliferation of mature β-cells, Wang et al. combined DYRK1A with pharmacological inhibition of transforming growth factor β superfamily (TGFbSF) / SMAD signal transduction, resulting in a significant further synergistic increase in human β-cell proliferation (average labeling index, 5%-8% and up to 15%-18%) and an increase in the number of β-cells in both mice and humans. In order to induce the proliferation of mature β-cells, intact islets were used and cells were not produced outside the islets. The cells do not move and remain in the islet structure. In order to release cells from the islets, 0.05% trypsin neutralized by adding an equal amount of 100% FBS was used to dissociate the islets. Although this work appears promising, the levels of proliferation observed are insufficient to generate the numbers of insulin-producing cells necessary to provide a viable source of insulin-producing cells for therapeutic use, such as for the treatment of diabetes.

[0012] The two standard techniques currently used to generate true beta cells as a source for cell therapy or transplantation in diabetes are highly intensive, requiring multiple steps, large amounts of factors and requiring many weeks to produce the desired cells. Cell-based therapies offer the prospect of treating and altering the course of diseases that are not adequately addressed by existing drugs, such as type 1 diabetes. Summary of the invention

[0013] The compositions and methods described herein overcome the problems of the above methods. Activated islet proliferating cell (AIPC) populations and methods for preparing them are disclosed herein. The AIPC population is capable of producing insulin and enables the expansion of the islet population to meet the requirements for use in cell-based therapy.

[0014] The methods described herein provide a rapid way to differentiate isolated islets into a population of AIPCs having a specific percentage of insulin-producing islets (which are denoted herein as "triple positive" islets) that have both CD133 cell markers and Ki-67-markers (described further herein). The so-called "triple positive" islets are glucose responsive and secrete both insulin and glucagon in response to appropriate stimuli.

[0015] A representative culture of the AIPC has been deposited with the ATCC under the terms of the Budapest Treaty [Accession No. PTA-125942].

[0016] Also disclosed herein are methods of treatment comprising administering to a mammal, eg, a human, an effective amount of a population of AIPCs to restore healthy pancreatic islets capable of producing insulin in response to glucose stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A and Figure 1B Microscope images of cultured rat islets (as further described herein, see Table 2) treated with cell culture medium containing IMG (≈3 μg / mL) are shown compared to rat islets cultured in basal medium alone (see Table 3). Within 24 hours after treatment, the treated islets became less clustered and the islet cells showed active migration away from the clusters. Control islets maintained their clustered morphology and showed minimal signs of cell migration. At 72 hours in culture, the treated islets had lost most of their morphology and began to establish colonies of insulin producing CD133-positive, Ki-67-positive proliferating islet cells. (See Example 1)

[0018] Figure 2A and Figure 2B Microscope images of cultured human islets treated with cell culture medium containing IMG (≈3 μg / mL) compared to untreated control islets are shown. As shown by rat islets (shown in Figure 1), cultured human islets treated with cell culture medium containing active agents showed the same trend of islet cell migration from islets (Day 2) and expansion of migrated islet cells into insulin producing CD133-positive, Ki-67-positive proliferating islet cells (Day 7). (See Example 2)

[0019] Figure 3 Shown is a bar graph comparing cell viability at days 3 and 10 as determined by propidium iodide and acridine orange staining of control cell populations (untreated mouse islet cells) versus treated cell populations followed by FAC; both populations (control / untreated and treated cells) exhibited over 80% cell viability at days 3 and 10. (See Example 1)

[0020] Figure 4A and Figure 4BA bar graph summarizing the results depicting the effects of culture medium containing active agents following culture with isolated human islets is shown; human islets cultured in culture medium containing IMG (≈10 μg / mL) resulted in an increase in islet cell number compared to controls (untreated human islets), as determined by the CyQUANT cell proliferation assay (which measures the DNA content of the cells). The assay was performed 3 days after islet plating, and cell counts were performed by trypan blue exclusion after the cells had been cultured in culture medium for 10 days to determine cell proliferation. Both cell measurements (cell proliferation and cell counts) showed a significant increase in the number of islet cells derived from human islets cultured in culture medium containing active agents, specifically, 6,400 cells in the untreated control compared to 9,500 cells after treatment with IMG ( Figure 4A ); and less than 500,000 cells in untreated controls, compared to approximately 2,500,000 cells after treatment with IMG ( Figure 4B ).

[0021] Figure 5 AIPCs generated by treating isolated, non-native islets with medium containing an active agent (IMG) were shown to be able to expand over at least two (2) passages within five (5) days. AIPCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then expressed using CellTrace TM CFSE Cell Proliferation Kit, analysis is performed 5 days after treatment with medium containing active agents. This kit is used for in vitro and in vivo labeling of cells to track multiple generations by flow cytometry using dye dilutions. Within 5 days, 2 generations of AIPC can be observed.

[0022] Fig. 6A and Figure 6B It is shown that isolated islets cultured in a medium containing an active agent (IMG) promotes the expansion of islets into a cell population (AIPC) containing an insulin, CD133 and Ki-67-positive cell phenotype compared to control islets. Specifically, at least 60% and greater than 70% (average) of the isolated islets cultured in a medium containing an active agent produced CD133 / Ki-67 / insulin positive AIPC (61.47%) (see Figure 6B ); however, control cells showed less than 5% of the control cell population as CD133 / Ki-67 / insulin positive (essentially 0%) (see Fig. 6A ). Using FACs, the expression of CD133 (a marker of somatic progenitor cells), Ki-67 (a nuclear protein associated with proliferation), and intracellular insulin expression were evaluated in cultured AIPCs. (See Examples 1 and 2).

[0023] Figure 7AIPC derived from isolated human islets secrete insulin after glucose stimulation after treatment with medium containing active agents. AIPCs were subjected to a static incubation glucose-stimulated secretion assay (see Example 2). Derived AIPCs were subjected to 5 stimulation conditions to evaluate insulin secretion from the medium for β-cell function. AIPCs were incubated for 30 minutes with the following: (1) 2.8 mM glucose (control baseline); (2) 16.7 mM glucose (for insulin stimulation); (3) 16.7 mM glucose + 100 μM IBMX (i.e., 3-isobutyl-1-methyl-xanthine, for maximum insulin stimulation); (4) 1.7 mM glucose + 1 μM epinephrine (for glucagon stimulation); (5) 5.6 mM glucose + 20 mM KCl (for other stimulations of insulin and glucagon). After the assay, the culture medium was stored at -20°C and subjected to standard ELISA analysis for insulin, wherein the results showed that for Group 1, the insulin secretion level was 11 μIU / mL insulin / million cells; for Group 2, 57 μIU / mL insulin / million cells; for Group 3, 221 μIU / mL insulin / million cells; for Group 4, 6 μIU / mL insulin / million cells; and for Group 5, 22 μIU / mL insulin / million cells.

[0024] Figure 8 AIPC derived from isolated human islets are shown to secrete glucagon after glucose stimulation after treatment with medium containing active agents. AIPC were subjected to a static incubation glucose stimulated secretion assay (see Example 2). Derived AIPC were subjected to 5 stimulation conditions to evaluate glucagon secretion from the culture medium for α-cell function. AIPC were incubated for 30 minutes with the following: (1) 2.8 mM glucose (control baseline); (2) 16.7 mM glucose (for insulin stimulation); (3) 16.7 mM glucose + 100 μM IBMX (for maximum insulin stimulation); (4) 1.7 mM glucose + 1 μM epinephrine (for glucagon stimulation); (5) 5.6 mM glucose + 20 mM KCl (for additional stimulation of insulin and glucagon). After the assay, the culture medium was stored at -20°C and subjected to standard ELISA analysis for glucagon, where the results showed that for Group 1, the glucagon secretion level was 32 pg glucagon / million cells; for Group 2, 30 pg / million cells; for Group 3, 52 pg / million cells; for Group 4, 98 pg / million cells; and for Group 5, 75 pg / million cells.

[0025] Fig. 9Bright field microscopy of derived AIPCs at 20 days in culture is shown, after which the derived AIPCs show signs that the AIPCs begin to generate secondary structures similar to islets (visible in the microscope and by staining). When tested, it was determined that these islet-like structures were diphenylthiocarbazone (DTZ) positive, representing putative β-cells in de novo constructed islets. DTZ binds to zinc ions present in islet β-cells and therefore stains the islets red (color results not shown).

[0026] Fig. 10A The FACs marker profile and morphology of derived AIPCs 60 days after treatment with medium containing an active agent (IMG) are shown. After 60 days in culture, the AIPC profile was evaluated (using flow cytometry as described in Example 2), and markers were evaluated as percentages (%) of cell types. The marker profile was as follows (as a percentage of cells): Insulin-positive / Ki-67-positive / CD133-positive showed 61.47% of the total cell population. About 10% of the cells were negative for the marker profile. In addition, Fig. 10B and Fig. 10C It was shown that derived AIPCs expanded according to the methods herein maintained their marker profile (CD133 / Ki-67 / insulin "triple" positive phenotype) and cell morphology for more than 60 days in culture.

[0027] Fig.11 It was shown that AIPCs remain viable in vivo and cause a decrease in blood glucose levels in a hyperglycemic mouse model (see Example 4). C57BL / 6J mice (n=6; indicated as M1, M2, M3, M4, M5, M6) were treated with STZ at a concentration of 75 mg / kg 4 days prior to AIPC infusion. On day 0, when the animals' fasting BG levels were greater than 200 mg / dL, each STZ-treated hyperglycemic mouse was injected with a cell suspension containing approximately four (4) million mouse AIPCs (isolated from tomato-red mice). The fasting blood glucose levels of each STZ-treated mouse were measured twice a week; two animals were sacrificed on day 28 after AIPC administration, and two animals were sacrificed on day 42. On day 46, the remaining 2 STZ-treated animals were injected with an additional (approximately) 10 million cells (each via the tail vein) and sacrificed on day 70. All STZ-treated animals that received AIPC injections showed an improvement in blood glucose levels, from 300-500 mg / dL before AIPC administration to approximately 200 mg / dL on day 50 after AIPC administration.

[0028] Fig.12Histological analysis of sacrificed animals (Example 4) shows donor tomato red AIPCs in the pancreas of STZ-treated animals, indicating migration / homing of tail-infused AIPCs to the pancreas via the bloodstream. AIPCs appear to be in greater concentrations for a longer period of time.

[0029] Fig.13 Immunohistological analysis of AIPC-infused mice sacrificed on day 70 (Example 4) showed areas within the pancreas that were "double positive" for both tomato red and Ki-67, indicating that AIPCs that had homed to the pancreas could divide and proliferate. DETAILED DESCRIPTION

[0030] Described herein are compositions and methods for promoting proliferation, expansion and differentiation of primary cell isolates containing pancreatic islets into endocrine progenitor cell populations containing cells positive for markers CD133 (stem cell marker) and Ki-67 (active proliferation marker), as well as insulin secretion. The composition comprises a culture medium containing a basal culture medium and an active agent, the active agent comprising an isolated polypeptide or an active fragment thereof according to (for example) SEQ ID NO: 1. Alternatively, the active agent may comprise an isolated polypeptide according to (for example) SEQ ID NO: 2-4. The method includes the use of the culture medium to promote differentiation and proliferation of endocrine progenitor cell populations containing marker profiles positive for CD1331 and Ki-67 from islet cells of isolated primary cells containing pancreatic islets.

[0031] Described herein are populations of endocrine progenitor cells (referred to as AIPCs, AIPCs, or AIPC populations), wherein the endocrine progenitor cell population is a) of mammalian origin; b) positive for the CD133 cell marker; c) positive for the Ki67 cell marker; d) produces -insulin in response to stimulation; e) produced by treating isolated primary cells containing pancreatic islets with a culture supplement comprising an active agent comprising a polypeptide or polypeptide fragment having an amino acid sequence identity according to (e.g.) SEQ ID NOs: 1-4.

[0032] Other methods include the use of endocrine progenitor cell populations generated by the methods described herein for cell transplantation or implantation, or for engineering tissue useful for treating a variety of pancreatic disorders or diseases.

[0033] The following terms are used in the present disclosure to describe different aspects of the present invention. These terms are used for the purpose of explanation only and are not intended to limit the scope of any aspect of the present invention.

[0034] As used herein, "active agent" refers to a protein, polypeptide, peptide fragment or analog thereof having an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with an amino acid sequence according to SEQ ID NO: 01, including SEQ ID NO: 2-4 (which represents a fragment of SEQ ID NO: 1), and includes any modifications thereof. In addition, peptide fragments or analogs thereof having an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with an amino acid sequence according to SEQ ID NO: 2-4, and include any modifications thereof.

[0035] As used herein, the term AIPC, AIPCs or AIPC population refers to a non-naturally derived endocrine (pancreatic) progenitor cell population produced according to the methods described herein, wherein the cell population is positive for the cell markers CD133 and Ki67 and produces insulin in response to stimulation.

[0036] As used herein, the term "growth" refers to the maintenance of cells in a living state, and may include, but is not limited to, proliferation and / or differentiation of cells. The term "proliferation" refers to an increase in the number of cells present in culture due to cell division.

[0037] As used herein, "culture", "cultured" or "culturing" refers to the removal or separation of cells from an environment (such as a host mammal) and their subsequent growth in a favorable in vitro artificial environment. "Cultured cells" are intended to include subculture (i.e., passaging) by transferring cells to a new container with fresh growth medium to provide more space for continued growth, differentiation and / or proliferation.

[0038] The term "expanded" is intended to mean that the resulting cell population is derived from ex vivo culture of islets in a vehicle composition comprising IMG. The term "expanded" should not be considered or limited to any mechanism or theory of cell origin and may include cells generated de novo in culture.

[0039] References to "pancreatic cells" include those cells normally present in the pancreas, and include pancreatic islet cells, e.g., glucagon-synthesizing α-cells, insulin-producing β-cells, and any combination thereof. AIPCs derived from pancreatic islet cells proliferated by the methods described herein are useful for the production of insulin and for implantation in subjects for the treatment of diabetes (including, e.g., type I diabetes), etc. Pancreatic cell populations are generally no more than 3% positive for Ki-67, a cell marker for identifying AIPCs generated from pancreatic cells using the methods described herein.

[0040] As used herein, the term "target" refers to an area in a recipient host (mammal, preferably human) that is in need of treatment or supplementation. The target can be a single area within a specific organ or can be multiple areas in the host. In some embodiments, whether or not the pancreas is targeted, the supplement or replacement results in the same physiological response as normal tissue, such as pancreatic tissue.

[0041] As used herein, the terms "treat," "cure," and "treating," and other grammatical equivalents as used herein, include alleviating, reducing, or ameliorating the symptoms of a disease or condition, preventing other symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., stopping the development of the disease or condition, alleviating the disease or condition, causing regression of the disease or condition, relieving the conditions caused by the disease or condition, or stopping the symptoms of the disease or condition, and preventing. These terms also include achieving a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit refers to the eradication or amelioration of the underlying condition being treated. Additionally, a therapeutic benefit is achieved by the eradication or amelioration of one or more physiological symptoms associated with the underlying condition, such that an improvement is observed in the patient, even though the patient may still be suffering from the underlying condition.

[0042] As used herein, "therapeutically effective amount" or "effective amount" means an amount sufficient to achieve the effect. The therapeutically effective amount for treating a condition is the amount of active substance that can achieve a clinically relevant endpoint in a patient or patient population. As a non-limiting example, the administration of an effective amount of a composition comprising AIPCs is an amount of about 4 million to about 14 million cells / kg, or an amount greater than 200 million cells, to reduce hyperglycemic subjects, such as patients with hyperglycemia, to a normal blood sugar range, about 100 to 125 mg / dL (5.6 to 6.9 mmol / L) to less than 150 mg / dL. The appropriate dose of AIPCs configured as a composition for injection or infusion will be based on the subject being treated and the severity of the condition being treated. The examples disclosed herein have been partially implemented in murine models and cell culture experiments using mammalian (human or murine) cells. Using a proportional method, such as an allometric proportional method, it is possible to predict a suitable and exemplary dosage range for the administration of a composition comprising AIPCs to an adult as disclosed herein. The dose ratio method is an experimental method and has been well identified and understood in the art. The method assumes that there are some unique features between species for anatomical, physiological and biochemical processes, and as such, possible differences in pharmacokinetic / physiological time are explained by the ratio method. As an example and not intended to be limiting, based on the literature, the human pancreas has 600,000 to 2,000,000 islets; therefore, there are about 600 million islet cells in the normal human pancreas, half of which are β-cells. Therefore, based on this scale, it is expected that a dose of 4,000,000 AIPCs / kg will be sufficient to replace the β-cells in the normal human pancreas.

[0043] 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 the sequences. Sequence identity can be measured as a percentage of identity; the higher the percentage, the higher the sequence identity. The percentage of identity is calculated for the entire length of the sequence. When aligned using standard methods, homologs or orthologous genes of amino acid sequences have a relatively high degree of sequence identity. When orthologous proteins are derived from more closely related species (e.g., human and mouse sequences), this homology is more obvious than less closely related species (e.g., human and C. elegans sequences).

[0044] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: 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; Huang et al. Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al., Meth. Mol.Bio.24:307-31,1994; Altschul et al., J.Mol.Biol.215:403-10,1990, which provides detailed considerations of sequence comparison methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) can be used to determine sequence identity levels (Altschul et al., J.Mol.Biol.215:403-10,1990), which is available from several sources, including the National Center for Biological Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md.20894, US) and the Internet.

[0045] The active agent may be a polypeptide comprising a 293 amino acid peptide, a peptide fragment thereof, such as a peptide fragment described in U.S. Patent Application No. 15 / 811,060, or a combination thereof; wherein the sequence of the polypeptide or fragment comprises at least 50% homology to a portion of the (293) amino acid sequence shown below and represented as SEQ ID NOs: 1-4:

[0046] Table I: SEQ ID NO: 1-4:

[0047] SEQ ID NO: 01 (293 aa; approximately 31 kDa fragment)

[0048]

[0049] SEQ ID NO: 2 (159 aa; approximately 17 kDa fragment)

[0050]

[0051] SEQ ID NO: 3 (75 aa; approximately 8 kDa fragment)

[0052]

[0053] SEQ ID NO: 4 (87 aa; approximately 9 kDa fragment)

[0054]

[0055] The amino acid residues of the active agent can be post-translationally modified or conjugated to other functional or non-functional molecular groups. See, for example, Guo et al. Mol. Biosyst. 7 (7): 2286-2295, 2011, which generally describes the antagonistic citrullination and methylation of human ribosomal protein S2 (e.g., SEQ ID NO: 1). Naturally, these modified amino acid residues are included in the amino acid sequence and within the scope of the active agent described herein.

[0056] Polypeptides and / or polypeptide fragments according to, for example, SEQ ID NOs: 1-4 can 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 / 811,060.

[0057] The first aspect relates to a composition for stimulating the growth, proliferation and differentiation of a pancreatic islet population into an AIPC population, wherein the composition comprises a culture medium containing a basal culture medium and an effective amount of an active agent, wherein the active agent comprises a polypeptide or an active fragment thereof comprising an amino acid sequence according to one or more of SEQ ID NOs: 1-4 (listed in Table 1). In one embodiment, the polypeptide comprises an amino acid sequence having at least 50% sequence identity with the amino acid sequence shown 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 the amino acid sequence shown in SEQ ID NO: 01. Alternatively, the polypeptide comprises an amino acid sequence having at least 50% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 2-4; in another embodiment, the polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 2-4.

[0058] A second aspect relates to a method for producing an AIPC population, comprising culturing a population of pancreatic islets in vitro with a cell culture medium comprising a basal medium and an effective amount of an active agent. The composition can be analyzed to determine the responsiveness of the composition to glucose, such as the ability to secrete glucose when exposed to a certain amount of glucose. The characteristics of the AIPC population relate to the ability to secrete glucagon when exposed to an appropriate stimulus.

[0059] A third aspect relates to a composition comprising a population of activated islet proliferating cells (AIPCs), wherein 60% of the AIPC population consists of CD133 cell markers and Ki-67 cell markers, and wherein at least 60% of the AIPC population is capable of producing insulin.

[0060] A fifth aspect relates to a composition comprising a population of activated islet proliferating cells (AIPCs) obtained by a method comprising culturing the islet population in vitro with a cell culture medium comprising a basal medium and an effective amount of an active agent.

[0061] The sixth aspect relates to a method for producing an AIPC population, comprising culturing an islet population in vitro with a cell culture medium comprising a basal culture medium and an effective amount of an active agent to obtain an AIPC population; screening an AIPC population for one or more cell markers that are selective for CD133, Ki-67, and insulin; and collecting an AIPC population identified by screening as CD133 / Ki-67-positive and insulin-produced from a cultured cell population. The method for producing an AIPC population may also include plating the AIPC population on a suitable culture medium, treating the cells with a culture medium comprising a basal culture medium and an effective amount of an active agent to obtain a cultured AIPC population, and passage the cultured AIPC until a suitable percentage of CD133 / Ki-67-positive and insulin-producing cells are proliferated. In addition, CD133+ / Ki-67+ / insulin+ ("triple positive") islet cells may be packaged, formulated, or encapsulated for delivery or transplantation to a mammal (e.g., human).

[0062] A seventh aspect relates to a method for amplifying and proliferating an activated islet proliferating cell ("AIPC") population, comprising enzymatically separating a cultured AIPC population to obtain an isolated AIPC population, and re-plating a composition comprising an isolated AIPC population and a cell culture medium comprising a culture medium containing a basal culture medium and an active agent onto a culture plate. Suitable proteases, such as trypsin, may be used in the amplification and proliferation of the AIPCs. Once separated, the AIPCs may be centrifuged at a speed (e.g., 1000 rpm) and duration (e.g., 7 min) sufficient to form particles. It may be desirable to centrifuge the AIPCs at a suitable cell density, e.g., about 1000 cells / cm 2To re-plate the isolated AIPCs. A characteristic of interest for AIPCs, including AIPC populations, isolated AIPCs, etc., is viability for at least 100 days after isolation.

[0063] The eighth aspect relates to a population of activated islet proliferating cells ("AIPC") in a packaged or microencapsulated formulation for administration or implantation into a mammal for in vivo therapy, specifically for the treatment of one or more pancreatic disorders, specifically type I diabetes. The AIPC population can be packaged as a delivery solution, or packaged in a delivery vehicle, and administered by implantation, injection, or infusion, whether the administration is systemic, local, or targeted.

[0064] The ninth aspect relates to a method for treating a pancreatic disorder, wherein the pancreatic disorder is hyperglycemia or type I diabetes in a mammal, comprising: culturing a population of pancreatic islets from a mammalian species in vitro in a culture medium comprising a basal culture medium and an effective amount of an active agent to obtain an AIPC population. In one embodiment, the AIPC population consists of insulin-producing CD133 / Ki-67-positive cells. In another embodiment, the method for treating a pancreatic disorder further comprises measuring the response of the AIPC population to glucose. In another embodiment, the method for treating a pancreatic disorder further comprises amplifying and proliferating the AIPC population; and further comprising implanting a composition comprising the AIPC population (e.g., primarily an insulin-producing CD133 / Ki-67-positive cell population) into a mammal, delivering the composition to a target site, thereby providing treatment for pancreatic disease. In one embodiment, the composition can be delivered as an aqueous solution, suspension, encapsulation, microencapsulation and / or encapsulated formulation or semisolid formulation; wherein the composition can be delivered to a target site in a mammal by one or more of injection, infusion, omental or peritoneal pocket, surgical implantation, or by packaging the composition as part of a device.

[0065] In another aspect of the present invention, the composition comprising a population of activated islet proliferating cells (AIPC) further comprises one or more buffers, one or more pharmaceutically acceptable carriers, or one or more pharmaceutically acceptable additives.

[0066] The cell culture system described herein includes at least an initial, islet population; a cell culture growth substrate; and a culture medium comprising a basal medium and an effective amount of an active agent, wherein the active agent includes a polypeptide according to (for example) SEQ ID NO: 1-4 or a fragment thereof, wherein 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, 96% or more, 97% or more, 98% or more or 99% or more sequence identity.

[0067] Example

[0068] The following examples are provided by way of illustration and not by way of limitation of the subject matter claimed herein. Throughout the examples, the term "IMG" is used to denote a specific active agent, which is represented herein as SEQ ID NO: 1, with the proviso that another active agent may be used, e.g., SEQ ID NO: 2-4, as described and defined herein. The use of the term IMG and its amount does not imply any specific dosage or concentration of the active agent. For the examples, the additives / supplements and concentrations of the active agent in the culture medium are described in Table 2, while the control culture medium is listed in Table 3. Culture Conditions.

[0069] The cell cultures performed in the examples were incubated at 37°C, standard CO2 (5%) conditions; the cultures (plating of cells, splitting) were performed under standard sterile techniques and in a vertical laminar flow clean bench and using the techniques and conditions described. Unless otherwise stated, AIPCs were cultured in the culture media described in the table. AIPCs were split when they reached about 70-80% confluence in culture.

[0070] The segmentation technique includes removing the supernatant from the culture plate (retaining the supernatant). Then, wash the plate with 2-5mL PBS (retaining the washing solution). Use about 3-5mL trypsin (such as obtained from Sigma-Aldrich) to separate AIPC by cultivating the cells at 37°C for about 3-5 minutes in the presence of trypsin until the cells are separated. Then, wash the plate again with PBS. Then, centrifuge the trypsinized cells, the retained PBS washing solution and the collected cell culture supernatant at 4°C for 7 minutes at 1000rpm. Pour out the resulting supernatant, and resuspend the particles in 2mL PBS and centrifuge again. Then remove the supernatant, resuspend the particles in a culture medium containing IMG, and centrifuge at about 1000 cells / cm 2 The cells were re-plated at a density of 1.

[0071] Table 2 Culture medium

[0072]

[0073] Table 3 Control culture medium

[0074]

[0075] Example 1

[0076] In an exemplary study, 300 mouse islets were isolated from the pancreas of male BALB / c mice (such as obtained from Jackson Labs or Charles River) aged 8 to 10 weeks. The islets separated were cultured in a culture medium comprising CMRL culture medium (originally developed by Connaught Medical Research Laboratories, and currently obtained from Mediatech, etc.) supplemented with 10% fetal bovine serum, 2mmol L-glutamine, antibiotics and an additive comprising the IMG of about 3.0 μg / mL concentration. The cells processed were grown and compared with control cells, which were the same cells grown in the basal culture medium without IMG. Within 24 hours, based on a visual inspection with 10 times magnification, by a microscope (using a Leica optical microscope with a Lumenera camera, Infinity analysis software), it was observed that the cells grown in the cell culture medium comprising IMG became less clustered, and the islet cells cultured seemed to actively migrate away from the cell clusters. On the other hand, it was observed that the control islet cells maintained their cluster morphology and showed minimal signs of cell migration. After 3 days of treatment, the islet cells cultured in the culture medium containing IMG showed both free floating and adherent cells in culture. As determined by acridine orange (AO) and propidium iodide (PI) staining, free floating cells are 30% viable. AO is an intercalating dye that can penetrate both living cells and dead cells, while PI is a cell-impermeable fluorescent solution for excluding non-living cells in flow cytometry analysis. PI is bound to double-stranded DNA by inserting between substrate pairs, but PI will be excluded by cells with complete plasma membranes. Adherent cells are 85% viable. On the contrary, control islet cell culture does not show free floating or adherent cell characteristics, although islet clusters seem to be healthy, as measured by cell viability>90%. By the 10th day, it has been shown that islet cells cultured in the culture medium containing IMG show low islet cell cluster percentages, there are no discernible free floating cells retained in culture, and adherent cells seem to form colonies. The total cell viability of the islet cells cultured in the culture medium containing IMG measured was greater than 90%. Although the control cells continued to show no migration or adhesion, the islet cluster cell viability remained at about 90%. At 25 days in culture, the islet cells from the treatment and control (untreated) cultures were fixed and stained for CD133 (somatic progenitor cell marker) and insulin. Immunohistochemical staining showed that the islet cells treated with the cell culture medium containing IMG were at least 75% insulin-positive. In addition, the islet cells treated were at least 70% CD133-positive; about 60% or more of the tested islet cells treated were positive for CD133 and insulin (considered to be "double positive").In contrast, control cells, although 90% insulin positive, showed positivity for the marker CD 133 in only less than 5% of the cell population. Likewise, less than 5% of the cell population was found to be doubly positive for both the CD 133 marker and insulin.

[0077] The results show that the cell culture system and culture medium described herein have the effect of inducing islet cell activation and migration from the islets. These cells form colonies of CD133 / insulin "double positive" cell populations, which presumably represent activated β-cell progenitors, which in turn can be used for methods and treatments for regenerating damaged islets.

[0078] Example 2

[0079] In another exemplary study, human pancreatic islets were used. It is the primary human islet processed from the organ donor pancreas, which is commercially available from Prodo Laboratories, Incorporated. Generally, the human islet population is less than 3% positive for Ki-67, and healthy islets respond to stimulation with about 50% insulin-production. In this example, ten (10) islets were cultured in a culture medium comprising CMRL 1066 culture medium, 10% fetal bovine serum, 10% human serum, 2mmol / L-glutamine, antibiotics, and IMG at a concentration of about 10.0μg / mL. The islets cultured in the culture medium comprising IMG were comparable to control cells cultured in the CMRL 1066 culture medium (alone) without IMG. The treated and untreated human islets were cultured at 37°C for 5 days, and then the CyQUANT cell proliferation assay (as obtained from Invitrogen) was performed. Within 5 days, human islets cultured in medium containing IMG showed approximately 1.5 times the number of cells of the control group: 6363 cells / mL in the control vs. 9469 cells / mL in islets cultured with IMG (p<0.001). Human islets cultured in medium containing IMG showed the same trends observed by microscopy in rat islets (Example 1), specifically, islet size became smaller, showed less organizational structure, and in time, islet cells appeared to actively migrate away from the cluster.

[0080] Example 3

[0081] Separately, human islets (3,000-10,000) are cultured in a control culture medium or a culture medium containing CMRL 1066 culture medium and supplemented with 10% fetal bovine serum, 10% human serum, 2mmol / L-glutamine, antibiotics and 10.0μg / ml concentration of IMG, on a plate coated with an adhesion factor mixture (AFM) comprising type I collagen (collagen from rat tail, Sigma-Aldrich C3867) and endothelial cell adhesion factor (ECAF, Sigma-Aldrich E9765). Different ratios of ECAF and collagen can be used, including collagen and ECAF in a 50 / 50 ratio. A thin layer of AFM (between 3-10mL) is applied and after 30 minutes of setting, excess AFM is removed and the plate is dried in a clean bench for 45 minutes. Before use, the plate is cleaned with PBS to remove any potential contaminants. Cultured islets are incubated in AFM-treated flasks (plates may be used) in culture medium or control medium for 2 to 5 days.

[0082] Based on the observed results using bright field microscopy, control islets (cultivated in standard CMRL culture medium without IMG) show that their cluster morphology is maintained, and do not show signs of cell migration.The expression of CD133 and Ki-67 (nuclear protein associated with proliferation) of islet culture (whether cultivated in the culture medium comprising IMG or cultivated in standard culture medium) was determined, and using BD FACSAria flow cytometer, intracellular insulin expression was determined by fluorescence activated cell sorting (FACS).Initially, CD133 expression marker culture cells were cultured, then according to the manufacturer's instructions, FOXP3 was fixed / permeabilized buffer, cells were fixed and permeabilized, and Ki-67 and intracellular insulin were dyed with conjugated fluorescent antibodies respectively.Then, after FOXP3 was fixed / permeabilized and dyed with conjugated fluorescent antibodies, FACS analysis was performed. It was found that human pancreatic islet cells cultured in medium containing IMG were positive for CD133, Ki-67 and insulin (triple positive), specifically: greater than 80% positive for insulin, CD133 and Ki-67, while control cells showed greater than 75% positivity for insulin and approximately 10-15% positivity for CD133 and Ki-67.

[0083] To test the glucose responsiveness of cultured islets, AIPCs were subjected to a static incubation glucose-stimulated secretion assay. 6Cells / well were plated in 6-well dishes and subjected to 5 stimulation conditions to evaluate insulin secretion from the culture medium for β-cell function and glucagon for α-cell function. Cells were incubated for 30 minutes with the following: (1) KREB buffer solution supplemented with physiological concentrations of 2.8 mM glucose (control baseline); 16.7 mM glucose (for insulin stimulation); (3) 16.7 mM glucose + 100 μM IBMX (for maximal insulin stimulation); (4) 1.7 mM glucose + 1 μM epinephrine (for glucagon stimulation); (5) 5.6 mM glucose + 20 mM KCl (for additional stimulation of insulin and glucagon). After the assay, the culture medium was stored at -20°C and subjected to standard ELISA analysis for insulin and glucagon. Islets cultured in cell culture medium containing IMG have been shown to secrete insulin in response to glucose stimulation, as shown in Figure 2. Figure 7 and Figure 8 shown.

[0084] Example 4

[0085] In order to determine whether AIPC is viable in vivo and to verify AIPC as a method for hyperglycemia treatment and glycemic control, 6 C57BL / 6J mice (Jackson Labs, product No 000664) were treated with streptozotocin (STZ) (75 mg / kg, a chemical used to destroy insulin-producing cells in mice and to produce type 1 diabetes phenotypes). Once STZ-treated animals (n = 6) showed fasting blood glucose levels above 200 mg / dL, (4 days after STZ treatment), phosphate buffered saline (PBS) water suspension or a cell suspension containing about 4 million AIPCs in 300 μL PBS was administered to STZ-treated mice by tail vein injection. AIPCs used in this study were isolated from Tomato-Red mice (Jackson Labs, product No 007576), whose cells expressed robust td-Tomato fluorescence.

[0086] To generate AIPCs for cell suspensions, mouse islets were isolated from tomato red mice by type 5 collagenase digestion (obtained from Sigma-Aldrich) according to previously published methods (Bertera et al., J Transplant. 2012; 2012: 856386, 2012 Dec 9). Briefly; 2-3 mL of cold collagenase solution (1.95 mg / ml in Hank's balanced salt solution (HBSS)) was injected into the pancreas through the common bile duct immediately after sacrifice. The fully expanded pancreas was removed and incubated at 37° C. for 20 minutes in a tissue culture flask, and then shaken for 5 seconds to break up the tissue. The digested tissue was washed with cold HBSS supplemented with 0.2% BSA, and the islets were purified by Ficoll gradient. In culture medium (Table 2), hand-picked islets were cultured at 37° C. under standard CO2 (5%) conditions. Prior to use as disclosed herein, T-75 tissue culture plates were pretreated with AFM (ECAF and type I collagen). For each T-75, approximately 300-500 islets were plated in culture medium (Table 2). Islets began to adhere to the plate, with cells migrating out as early as 24 hours after plating; by 72 hours, most islets adhered and cells migrated out; and at approximately 7-10 days, AIPCs converged. By the 14th day, AIPCs underwent serial passage. Then, cultured AIPCs were collected for injection into STZ-treated mice.

[0087] Fasting blood glucose levels of STZ-treated mice after AIPC administration were measured twice a week; 2 animals (M2 and M6) were sacrificed on day 28, and 2 animals (M3 and M5) were sacrificed on day 42 after AIPC injection. On day 46, an additional 10 million cells / 300 μL in suspension (PBS) were administered to the remaining 2 animals (M1 and M4) by tail vein injection and sacrificed on day 70 of the study.

[0088] The results showed that STZ-treated AIPC-injected mice showed reduced fasting blood glucose levels, which decreased over time. Finally, two animals (those STZ-treated mice that received two AIPC injections, receiving a total of about 14 million cells) showed normoglycemia (blood glucose levels below 200 mg / dL) on day 53 (see Fig.11 Furthermore, histological and immunological analyses demonstrated that AIPCs injected into STZ-treated mice via the tail vein homed to or engrafted into the pancreas and, once engrafted into the pancreas, continued to proliferate, as shown by histological staining and microscopic analysis (in Fig.12 and Fig.13 Results are shown).

[0089] Histological analysis of sacrificed animals showed donor tomato red AIPCs in the pancreas of STZ-treated animals, indicating migration / homing of tail vein infused AIPCs to the pancreas (see Fig.12 ). AIPCs appear to be in greater concentrations for a longer period of time. Immunohistological analysis of STZ-treated mice that received AIPCs by tail vein injection and were subsequently sacrificed at day 70 showed areas within pancreatic tissue that were doubly positive for both tomato red and Ki-67 (based on histological staining for the markers), indicating that the injected AIPCs homed to the pancreas where they were able to continue cell division and proliferation (see Fig.13 ).

[0090] Method described herein is included in the step of cultivating the islet of pancreas that separates in the cell culture medium that comprises basal medium and activating agent.Culture medium does not show the sign that causes islet cell viability to reduce, and shows the direct sign that promotes islet dissociation and islet cell to move from the inner core of islet of pancreas.During treatment with culture medium, the amplification of the islet cell of this culture that produces has been shown.The produced cell that expands in the cell culture medium that comprises activating agent is positive to insulin expression, CD133 and Ki-67 test, and represents the novel AIPC cell colony that presumes to use method described herein and culture medium to produce.The AIPC that will be produced by the culture of islet of pancreas that separates and comprises activating agent is passed on to posterity continuously for more than 18 generations, and show to remain on and survive at least 100 days after separation.

[0091] The derived activated islet proliferating cell population produced according to the methods herein comprises at least 30% CD133+ / Ki-67+ cells in culture; or comprises at least 35% CD133+ / Ki-67+ cells in culture; or comprises at least 40% CD133+ / Ki-67+ cells in culture; or comprises at least 45% CD133+ / Ki-67+ cells in culture; or comprises at least 50% CD133+ / Ki-67+ cells in culture; or comprises at least 50% CD133+ / Ki-67+ cells in culture. or at least 80% of CD133+ / Ki-67+ cells in culture; or at least 85% of CD133+ / Ki-67+ cells in culture; or at least 90% of CD133+ / Ki-67+ cells in culture; and wherein at least 60% of the derived activated islet proliferating cells are also insulin producing.

[0092] Features of encapsulation and implantation of in vitro produced islet cells into mammals have been previously described in the art (see, e.g., Altman et al., 1984, Trans. Am. Soc. Art. Organs 30:382-386 and U.S. Pat. No. 6,703,017 B1, incorporated herein by reference) - and they will be suitable for AIPCs produced according to the methods disclosed herein. Preferably, the encapsulating agent is hypoallergenic, easily and stably localized in the target tissue, and provides additional protection for the implanted cell composition to protect and prevent destruction of the implanted IPPCs.

[0093] Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent that certain changes and modifications may be practiced within the scope of the appended claims. It will be apparent to those skilled in the art that the features described with respect to any aspect and multiple embodiments as described above may be interchangeably applicable between different embodiments.

[0094] Aspects and embodiments of the present invention as described above are examples describing the various features of the present invention. All patent disclosures and patent applications mentioned in this application represent the level of a person skilled in the art to which the present invention belongs. All patent disclosures and patent applications are incorporated herein by reference to the same extent that each individual patent disclosure or patent application specifically and individually indicates that it is incorporated by reference.

[0095] Throughout the description and claims of this specification, the words "comprise" and "contain" and their variations mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, when the indefinite article is used, the specification should be understood to contemplate plurality as well as singularity unless the context requires otherwise.

[0096] The characteristics, features, compounds, chemical moieties or groups described in conjunction with a specific aspect, embodiment or example of the invention will be understood to apply to any other aspect, embodiment or example described herein unless contradicted therewith. All characteristics disclosed in this specification (including any attached claims, abstracts and drawings) and / or all steps of any method or process disclosed thereby may be combined in any combination, except for combinations in which at least some of these characteristics and / or steps are mutually exclusive. The present invention is not limited to the details of any of the above embodiments. The present invention extends to any novel characteristic or any combination of novel characteristics of the characteristics disclosed in this specification (including any attached claims, abstracts and drawings), or to any novel step or any combination of novel steps of the steps of any method or process disclosed thereby.

[0097] The information disclosed herein is incorporated herein by reference, including, for example, U.S. Patent Application No. 15 / 811,060, filed on November 13, 2017; PCT / GB2019 / 050049, filed on January 9, 2019; and U.S. Provisional Patent Application No. 62 / 689,780, filed on June 25, 2018. In the event of a conflict between a term and / or expression introduced herein and a term and / or expression disclosed herein, the information disclosed herein shall prevail.

Claims

1. A composition for stimulating the growth, proliferation and differentiation of a pancreatic islet population into an activated pancreatic islet proliferating cell population, wherein the composition comprises a culture medium containing a basal culture medium and an effective amount of an active agent, wherein the active agent comprises a polypeptide consisting of an amino acid sequence corresponding to SEQ ID NO:

1.

2. The composition according to claim 1, wherein the basal medium comprises one or more of an antibiotic agent, an antifungal agent, albumin and L-glutamine.

3. The composition according to claim 1 or 2, wherein the polypeptide consisting of the amino acid sequence corresponding to SEQ ID NO: 1 is present in a concentration ranging from 1-20 μg / mL.

Images