Compositions and methods for expanding insulin- and glucagon-secreting cells from pancreatic tissue of type 1 diabetic patients and therapeutic uses thereof

Abstract

Disclosed herein are compositions containing cell-based therapeutics useful for the treatment of pancreatic disorders, including type 1 diabetes, and methods for producing the compositions.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 63 / 247,252, filed September 22, 2021, and 63 / 337,137, filed May 1, 2022, the contents of which are incorporated by reference in their entireties. [Background technology]

[0002] Although cell-based therapies hold the promise of transforming the treatment and course of pancreatic diseases such as type 1 diabetes (T1D) that are not adequately addressed by existing therapies, cell-based therapies face a myriad of issues, primarily related to safety and efficacy, as well as scalability of manufacturing. Many of the issues associated with cell-based therapies are described in Engineering the next generation of cell-based therapeutics by Bashor, CJ, et al., Nat Rev Drug Discov (2022) (available online at https: / / doi.org / 10.1038 / s41573-022-00476-6). Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein are compositions comprising cell-based therapeutics and methods of producing compositions useful for the treatment of pancreatic disorders, including type 1 diabetes. In one embodiment, compositions as disclosed herein comprise insulin and glucagon secreting cell populations generated from non-insulin secreting pancreatic cells obtained by needle biopsy from a type 1 diabetic donor pancreas. In another embodiment, compositions disclosed herein comprise insulin and glucagon secreting cell populations generated from pancreatic cells obtained by needle biopsy from a patient or donor suffering from chronic pancreatitis. In one embodiment, non-insulin secreting type 1 diabetic pancreatic cells are treated in vitro with an islet cell culture medium comprising a basal medium and an effective amount of a polypeptide according to the amino acid sequence set forth in SEQ ID 1 or 2, and the treated cells differentiate and proliferate into a population of islet progenitor cells that secrete both insulin and glucagon in response to stimulation and are CD133 positive. The resulting insulin and glucagon secreting progenitor cells can be expanded to a desired cell number for subsequent use in transplantation or injection and as a cell-based therapeutic for type 1 diabetes or chronic pancreatitis. A cell composition comprising an effective amount of a population of insulin and glucagon-secreting progenitor cells can be administered to a subject by infusion, injection, implantation, intraportal administration, or other suitable delivery means, such as with a medical device, as a method of restoring insulin and glucagon secretion in response to stimulation.

[0004] The compositions and methods disclosed herein have implications for cell-based therapy and cell transplantation, i.e., for producing large amounts of insulin- and glucagon-secreting pancreatic cells useful for autologous or allogeneic transplantation for the treatment of type 1 diabetes or chronic pancreatitis.

[0005] Also disclosed herein is a method of treating a pancreatic disorder, such as type 1 diabetes or pancreatitis, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an insulin- and glucagon-secreting pancreatic cell population, where the insulin- and glucagon-secreting pancreatic cell population is generated by treating pancreatic cells obtained, such as via needle biopsy, from diseased pancreatic tissue (e.g., a type 1 diabetes subject or one suffering from chronic pancreatitis) with an islet cell culture medium comprising a basal medium and a peptide comprising an amino acid sequence spanning sequence ID 1 or 2. The composition, when administered to a subject in need thereof, provides for delivery of healthy pancreatic progenitor cells to a target site in the subject, where the healthy pancreatic progenitor cells are capable of producing insulin and glucagon in response to stimulation.

[0006] Compositions comprising therapeutically effective amounts of insulin- and glucagon-secreting progenitor cells produced by the methods disclosed herein can be used as autologous or allogeneic cell-based therapy to compensate for loss of insulin production or to replace insulin production in subjects with type 1 diabetes, or other diseases characterized by severe insulin deficiency, such as after total or partial pancreatectomy, with or without autologous or allogeneic islet transplantation.

[0007] In one embodiment, the composition may be prepared for transplantation by supplementing the composition with human serum albumin and / or human serum from the recipient prior to administration.

[0008] In another embodiment, a pancreatic islet cell culture medium useful for stimulating the growth, proliferation and differentiation of insulin- and glucagon-secreting cells from pancreatic cells derived from pancreatic tissue of a type 1 diabetic patient comprises a basal medium and an effective amount of a polypeptide, said polypeptide comprising an amino acid sequence according to one or more of SEQ ID NOs: 1-2 (as set forth in Table 1), or an active fragment thereof. In one embodiment, the polypeptide comprises an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth 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 to the amino acid sequence set forth in SEQ ID NO: 1. Alternatively, the polypeptide comprises an amino acid sequence having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and 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 set forth in SEQ ID NO:2.

[0009] In yet another embodiment, the cell composition comprises a population of insulin and glucagon secreting cells generated by treating isolated type 1 diabetic pancreatic tissue with a pancreatic islet cell culture medium comprising a base medium and an effective amount of a polypeptide according to SEQ ID NO: 1 or 2 or an active fragment thereof, further comprising measuring the response of the cells to glucose, wherein the cell composition comprises a population of cells capable of secreting insulin and glucagon in response to an appropriate stimulus.

[0010] In another embodiment, a method for producing a cell composition includes applying in vitro a base medium and an effective amount of SEQ ID NO: 1 or 2, or an active fragment thereof, to human pancreatic tissue collected from a type 1 diabetes patient, culturing the cells in pancreatic islet cell culture medium, screening the cultured cells for one or more cell markers selective for CD133 and insulin, and collecting cells from the cultured cell population that are screened as CD133 and insulin positive, and continuing to grow the cultured cells until a desired amount of cells have grown.

[0011] In yet another embodiment, a cell composition comprising insulin and glucagon secreting cells derived from T1D pancreatic tissue is packaged or encapsulated for administration or implantation into a mammal for in vivo treatment, particularly to restore insulin production and secretion. The cell composition can be packaged as a delivery solution or in a delivery vehicle and administered by implantation, injection or infusion, whether the administration is systemic, local, or directed to a target site.

[0012] In yet another embodiment, a method of treating a pancreatic disorder, wherein the pancreatic disorder is characterized by insufficient production of insulin, comprising culturing in vitro in a mammal a population of insulin- and glucagon-secreting cells from pancreatic tissue harvested from a type 1 diabetic donor pancreas in an islet cell culture medium comprising a basal medium and an effective amount of a polypeptide set forth in SEQ ID NO: 1 or 2 or an active fragment thereof, thereby generating a population of CD133 positive, insulin- and glucagon-secreting cells, further comprising isolating and expanding the population to generate a predominant (at least 60% or more) population of insulin- and glucagon-secreting cells, and further comprising harvesting the insulin- and glucagon-secreting cells, suspending the harvested cells in a physiological buffer, such as phosphate buffered saline (PBS) or Hank's balanced salt solution (HBSS), and transplanting or injecting the cell composition comprising the insulin- and glucagon-secreting cells suspended in the physiological buffer into a mammal. In one embodiment, the composition may be delivered as an aqueous solution, suspension, encapsulated, microencapsulated, and / or encapsulated, or semi-solid formulation, where the composition is one or more of being administered to a target site in a mammal by injection, infusion, ovarian or peritoneal pouch, surgical implant, or by packaging the composition as part of a device.

[0013] In another embodiment, the cell composition comprises an insulin and glucagon secreting cell population further comprising one or more of a buffer, a pharma- ceutically acceptable carrier, a pharma-ceutically acceptable excipient, an antibiotic or other medicinal agent. [Brief description of the drawings]

[0014] The compositions and methods disclosed herein are further illustrated by the accompanying figures, in which the term "IPC" is used to refer to the insulin producing cells (IPCs) described and claimed herein. [Figure 1]FIG. 1 shows that T1D-derived insulin- and glucagon-secreting cells grown according to the methods herein are greater than 50% triple positive for CD133, insulin, and glucagon. [Diagram 2] We show that T1D pancreatic tissue cultured in islet cell culture medium containing peptides according to SEQ ID NO: 1 or 2 produces cells that secrete insulin in response to glucose stimulation as indicated by a stimulation index, which is the ratio of insulin secretion under high glucose conditions to basal secretion under unstimulated conditions. A value greater than 2 represents glucose responsiveness in the cells. Sample 1 is a cell population grown from normal pancreatic tissue according to the methods herein, and Samples 2-4 are cell populations grown from T1D pancreatic tissue according to the methods herein. [Diagram 3] The figure shows down- and up-regulation of genes (families) associated with pancreatic function in single T1D biopsy-derived cell preparations compared to native pancreatic tissue. Each gene family contains 5-13 genes. As the figure shows, cell populations consisting of insulin- and glucagon-secreting cells generated according to the methods herein show up-regulation of gene families essential for mature islet cells, β-cell maturation, GSIS, insulin granules, and cell cycle. [Figure 4] 1 shows serum insulin levels after transplantation of cell compositions containing insulin and glucagon grown according to the methods herein into streptozotocin (STZ, a beta cell specific toxin that induces irreversible damage to pancreatic islets and induces diabetes) treated mice. The cell compositions were shown to promote the secretion of human insulin in vivo, which was present in the serum of STZ mice treated with the cell compositions disclosed herein for up to 100 days. [Diagram 5] We show that insulin- and glucagon-secreting cells generated from type 1 diabetes (T1D) cells grown according to the methods herein can normalize blood glucose levels when injected in an STZ diabetic mouse model. M1-4 refer to the STZ mouse sample numbers, i.e., mouse-1, mouse-2, mouse-3, mouse-4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The following terms are used in this disclosure to describe different embodiments. These terms are used for descriptive purposes only and are not intended to limit the scope of any aspect of the subject matter claimed herein.

[0016] As used herein, "SEQ ID NO: 1 or 2" refers to a protein, polypeptide, peptide fragment, or analog thereof, and any variant thereof, having an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2 (see Table 1). It is also intended to include a peptide fragment, or analog thereof, and any variant thereof, having an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and 2. In vitro studies with the polypeptides set forth in SEQ ID NO: 1 and SEQ ID NO: 2 have demonstrated that treatment of cells (keratinocytes, intestinal cells, pancreatic islet cells, endothelial cells, and lung cells) with a polypeptide according to SEQ ID NO: 1 or SEQ ID NO: 2 added to cell culture medium results in stimulation and increased cell growth, as measured by the percentage of CD133 positive cells and MTT cell proliferation assay (Sigma Aldrich Cell Proliferation Kit), resulting in viable progenitor cells. These progenitor cells can regenerate and multiply by the billions.

[0017] As used herein, the terms "insulin and glucagon secreting cells" or "insulin and glucagon secreting pancreatic islet cells" or "insulin and glucagon secreting progenitor cells" are used interchangeably to refer to a cell composition comprising insulin and glucagon secreting cells and / or cell population(s), which are generated from non-insulin secreting T1D pancreatic cells according to the methods described herein, are positive for the cell markers: CD133, insulin, glucagon, and produce insulin and glucagon in response to stimulation, and are further characterized by the cell markers PDX-1, SST, IIAP, Pax4, Pax6, Nkx2, Nkx6, NeuroD1, MafA, MafB.

[0018] "Proliferation" refers to an increase in the number of cells present in culture as a result of cell division.

[0019] As used herein, "culture," "cultured," or "culturing" refers to the removal or separation of cells from an environment (such as within a host mammal) and their subsequent growth in vitro in a favorable artificial environment. "Cultured cells" is intended to include subculture (i.e., passaged) of cells by transferring them to a new container with fresh growth medium to provide more space for continued growth, differentiation, and / or proliferation. Reference to "pancreatic cells" includes cells normally found in the pancreas of a mammal, including pancreatic islet cells, e.g., glucagon-synthesizing alpha cells, insulin-producing beta cells, and any combination thereof.

[0020] The term "target site" as used herein refers to an area in a recipient host (mammal, preferably human) that requires treatment or replacement. The target site may be a single area in a particular organ, or may be multiple areas in the host. In some embodiments, the replacement or replacement results in the same physiological response as a normal tissue, such as pancreatic tissue, whether or not the pancreas is targeted.

[0021] As used herein, the terms "treat", "treating" or "treatment" and other grammatical equivalents include alleviating, reducing or ameliorating the symptoms of a disease or condition, preventing additional symptoms, improving or preventing the underlying metabolic causes of the symptoms, inhibiting a disease or condition, e.g., arresting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating conditions caused by a disease or condition, or arresting the symptoms of a disease or condition, and prevention. The term further includes achieving therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to the eradication or amelioration of the underlying disease being treated. Therapeutic benefit is also achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease, such that an improvement is observed in the patient, even though the patient may still be afflicted by the underlying disease.

[0022] As used herein, "effective amount" refers to an amount sufficient to achieve a described effect. A therapeutically effective amount for treating a condition is an amount capable of achieving a clinically relevant endpoint in a patient or patient population. As a non-limiting example, administration of an effective amount of a composition comprising insulin and glucagon secreting cells can produce enough insulin to lower blood glucose levels to about 100-125 mg / dl (5.6-6.9 mmol / L), or to less than 250 mg / dl, for example, about 1.2 to about 2.5 x 10 6 cells / kg, or 200 x 10 6 Other ranges include approximately 3×10 per kg body weight. 6 Cells ~ approx. 25×10 6 cells, or approximately 5 × 10 per kg of body weight 6 ~About 10×10 6Million cells. The appropriate dose of the composition depends on the subject being treated as well as the severity of the condition being treated, by route of administration such as injection or infusion or implantation. Scaling methods such as allometric scaling can be used to predict appropriate and exemplary dose ranges for administering compositions as disclosed herein to adult humans. Dose scaling is an empirical approach, well characterized and understood in the art. This approach assumes that there are some inherent properties regarding anatomical, physiological, and biochemical processes between species, and possible differences in pharmacokinetics / physiological times are, as such, to be accounted for by the scaling. By way of example, and not by way of limitation, based on the literature, the human pancreas contains 6×10 5 From about 2 × 10 6 , and thus, a normal human has approximately 600 × 10 6 10 islet cells, half of which are beta cells. Considering that 30% of the islet mass is sufficient to maintain normoglycemia, 1.2×10 islet cells of the insulin- and glucagon-secreting cells disclosed herein are 6 is expected to fully replace the insulin-producing capacity of the non-diseased human pancreas.

[0023] As used herein, the term "sequence identity" refers to the identity between two or more amino acid sequences, expressed as the identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity, with a higher percent meaning that the sequences are more identical. The percent identity is calculated over the entire length of the sequences. Homologs or orthologs of amino acid sequences possess relatively high sequence identity when aligned by standard methods. This homology is more pronounced when the ortholog proteins are from more closely related species (e.g., human and mouse sequences) compared to more distantly related species (e.g., human and nematode sequences). Methods for alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman; 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; Carpet 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. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed discussion of sequence alignment methods and homology calculations. The level of sequence identity can be determined using the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990), available from:There are several sources of information, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894, US) and the Internet.

[0024] It will be understood that numerical values ​​may be associated with a certain amount of experimental error. Thus, the recitation of the modifier "about" (or "approximately") before a numerical error is meant to embody the experimental error that may be associated with the recitation of the numerical value. To the extent that an experimentally obtained numerical value is not preceded by the term "about" (or "approximately"), it does not mean that the numerical value is not associated with a certain amount of experimental error.

[0025] Representative cultures of insulin- and glucagon-secreting cells characterized herein were deposited with the ATCC on September 7, 2022 [accession no. [Based on the Budapest Treaty] Cultured cells, expanded cells, isolated cells, etc. can be protected from external mutagenic stimuli, for example ultraviolet light.

[0026] Using the methods disclosed herein, it has been determined that 30 days after isolation of T1D pancreatic tissue, a single pancreas can produce 77 billion insulin- and glucagon-secreting (islet) cells, and 2 trillion by day 60, sufficient to infuse 100-150 patients in need of treatment (depending on severity and dosage) or bank (cryopreserve) cells for future expansion and reinfusion. [Table 1]

[0027] The amino acid residue of the active agent can be post-translationally modified or conjugated with other functional or non-functional molecular groups.For example, Guo et al.Mol.Biosyst.7(7):2286-2295,2011, describes the roughly opposing citrullination and methylation of human ribosomal protein S2 (e.g., SEQ ID NO:1).Of course, such modified amino acid residues are included in the amino acid sequence and are within the scope of the active agent described herein.

[0028] For example, the polypeptides and / or polypeptide fragments set forth in SEQ ID NOs: 1 and 2 can be produced under conditions known in the art for protein production, e.g., produced in bacteria, yeast, or by synthetic means, or as described in U.S. Patent Application Serial No. 15 / 811,060.

[0029] In one embodiment, the cell composition may be packaged as a delivery solution or in a delivery vehicle that constitutes a medical device, and may be administered by implantation, injection, or infusion, whether the administration is parenteral, systemic, local, or directed to a target site. In one embodiment, encapsulation and transplantation of in vitro generated insulin- and glucagon-secreting pancreatic islet cells into a mammal would be suitable for insulin- and glucagon-secreting cells previously characterized in the art (e.g., Altman, et al., 1984, Trans. Am. Soc. Art. Organs 30:382-386, and U.S. Patent No. 6,703,017 B1, incorporated herein by reference) and generated according to the methods disclosed herein. Preferably, the encapsulating agent is hypoallergenic, easily and stably located in the target tissue, and can add protective functions to the transplanted cell composition, protecting and preventing the destruction of the transplanted cells.

[0030] The appropriate graft dose in humans can be determined from existing information related to ex vivo islet transplantation in humans, further in vitro and animal studies, and human clinical trials. From data on ex vivo islet transplantation in humans, it is predicted that approximately 8,000 to 12,000 islets per kg of patient may be required. Assuming long-term survival of the implant after transplantation, this may be less than the number of naturally occurring islets (approximately 2 million in a normal human adult pancreas) or even less than the amount used in ex vivo islet transplantation.

[0031] In one embodiment, the cell composition has therapeutic benefit for treating a pancreatic disorder in a mammal, where the pancreatic disorder is hyperglycemia, type 1 diabetes, or chronic pancreatitis, comprising administering a therapeutically effective amount of an insulin- and glucagon-secreting cell population, thereby providing a treatment for the pancreatic disorder.

[0032] In one embodiment, a composition comprising a therapeutically effective amount of a population of insulin- and glucagon-secreting cells may be formulated as an aqueous solution, suspension, encapsulated, microencapsulated, and / or encapsulated, or semi-solid formulation, where the composition may be delivered to a patient in need via one or more of injection, infusion, cartilage or peritoneal pouch, port, surgical implant, or packaging the composition as part of a device and delivering it to a target site in a mammal.

[0033] In one embodiment, the composition comprises a population of insulin and glucagon secreting progenitor cells and further comprises one or more of a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable additive and / or a pharmaceutical agent. Suitable excipients and additives include, but are not limited to, buffers such as PBS or HBSS, amino acids, stabilizers or bulking agents, surfactants, antibacterial / antiseptic agents, antifungal agents, metal ions / chelators, polymers, polyanions, salts, sugars, cyclodextrin-based excipients, lyoprotectants, solubilizers, antioxidants, complexing agents, anti-adherents, dispersing agents, serum additives, and the like.

[0034] In another embodiment, the disclosure provides a method of treating a mammal, preferably a human, suffering from or at risk of developing type 1 diabetes or severe pancreatitis, comprising the steps of resecting pancreatic tissue from the mammal, culturing the resected pancreatic tissue in vitro to expand a population of insulin- and glucagon-secreting islet cells, and transplanting, implanting, injecting, or otherwise inserting the population of insulin- and glucagon-secreting islet cells, alone or in conjunction with a medical or delivery device, into the mammal.

[0035] Working Example The following examples are offered by way of illustration, but not by way of limitation, of the subject matter claimed herein.

[0036] The cell cultures performed in the examples were incubated at 37°C under standard CO (5%) conditions, and the cultures (cell plating, splitting) were performed in a vertical laminar flow hood using standard aseptic techniques and conditions. Unless otherwise noted, cells (including controls) were cultured in islet cell culture medium as described in Tables 2 and 3. Cells were split when they reached approximately 70-80% confluence.

[0037] In one embodiment, the splitting technique involved removing the supernatant from the culture plate (the supernatant was saved). The plate was then washed with 2-5 ml of PBS (the wash was saved). The cells were detached using about 3-5 ml of trypsin (available from Sigma-Aldrich) by incubating the cells in the presence of trypsin for about 3-5 minutes at 37° C. until the cells detached. The plate was then washed a second time with PBS. The trypsinized cells, the saved PBS wash, and the collected cell culture supernatant were centrifuged at 300 g for 7 minutes at 4° C.

[0038] In one embodiment, the resulting supernatant is decanted and the pellet is resuspended in 2 ml of PBS and recentrifuged. The supernatant is then removed and the pellet is resuspended in medium containing SEQ ID NO: 1 or 2 at ∼1000 cells / cm. 2The cells were replated at a cell density of 1000 ng / ml. The examples may refer to cell culture plates, but it will be understood that cell culture flasks are an acceptable alternative to plates. [Table 2] EXAMPLES

[0039] A method for generating insulin- and glucagon-secreting pancreatic cell populations from non-insulin-producing pancreatic tissue obtained by needle biopsy from type 1 diabetic donors. Human pancreatic tissue was obtained by needle biopsy from a donor patient with type 1 diabetes (T1D) (58-year-old female, with a 53-year history of diabetes). 1 × 1 mm pieces were cut from donor pancreases (obtained from the Organ Recovery Education Center) that had been stored on ice in a commercially available solution (sold under names such as Viaspan, Belzer UW, Bel-Gen, or StoreProtec). 3 A biopsy was obtained.

[0040] The harvested T1D tissue was then cultured in islet cell culture medium (see Table 2) containing L-glutamine (2 mmol), ciprofloxacin (2 mg / L), amphotericin B (0.1 mg / L), penicillin (100,000 units / L), and streptomycin (100,000 micrograms / L), as well as the polypeptide set forth in SEQ ID NO: 1 or 2 (in the range of 3-20 μg / ml, specifically 10 μg / ml), and CMRL (e.g., Mediatech #99-663-CV Transplant Medium (CMRL 1066) without phenol red) supplemented with fetal calf serum (FCS) (10%) and human serum (10%). Control medium consisted of CMRL and fetal calf serum (FCS) supplemented with L-glutamine (2 mmol), ciprofloxacin (2 mg / L), amphotericin B (0.1 mg / L), penicillin (100,000 units / L), and streptomycin (100,000 micrograms / L) (10%) and human serum (10%) (without the addition of polypeptides according to SEQ ID NO: 1 or 2). Standard tissue / cell culture conditions (37°C, 5% CO2) were used.

[0041] Tissues are cultured in plates or flasks coated with an attachment factor mixture (AFM) containing type I collagen (rat tail collagen, Sigma-Aldrich C3867) and endothelial cell attachment factor (ECAF, Sigma-Aldrich E9765). Various ratios of ECAF to collagen can be used, including but not limited to a 50 / 50 ratio of collagen to ECAF. Briefly, plates (or flasks) were prepared by applying a thin layer of AFM (between 3-10 ml) to the plate, allowed to set for 30 min, and then excess AFM was removed. Plates were allowed to dry for 45 min in a hood. Prior to use, plates were washed with PBS to remove potential contaminants. Harvested tissues were incubated on AFM-treated plates in islet cell culture medium supplemented with the polypeptides set forth in Sequence ID No: 1 or 2 until cells began to mobilize and proliferate (approximately 10-20 days).

[0042] After 12-15 days in culture, the biopsy-derived cells mobilized, attached to the dish, and began to proliferate. Over the next 4-6 weeks, the attached cells continued to proliferate, becoming confluent and doubling every 3 days. The biopsy-derived cells in culture showed morphological similarities to a cell composition consisting of insulin-secreting cells generated from a nondiabetic donor previously characterized in US2021 / 0205371 published July 8, 2021. Similarly, the cells formed islet-like cell clusters with a size consistent with that of islets of Langerhans and were shown to secrete insulin in response to stimulation with glucose.

[0043] The resulting cell cultures from T1D pancreatic biopsy tissues were also assayed for CD133 expression, as well as intracellular insulin and glucagon expression, by fluorescence-activated cell sorting (FACS) using a flow cytometry device (Becton Dickinson FACS Aria cell sorter). Cultured cells were first labeled for CD133 expression, then fixed and permeabilized with FOXP3 fixation / permeabilization buffer and stained with fluorescent antibodies conjugated for glucagon and intracellular insulin, respectively, according to the manufacturer's instructions. FACS analysis was performed following FOXP3 fixation / permeabilization and staining with fluorescent antibodies conjugated for glucagon and intracellular insulin, respectively. T1D pancreatic biopsy-derived cells cultured in islet cell culture medium containing the peptides set forth in SEQ ID NO:1 or 2 were found to be positive for CD133, glucagon and insulin (referred to herein as "triple positive"), specifically, 48-73% triple positive for insulin, glucagon and CD133, 26-42% double positive for glucagon and CD133, 14-18% triple negative for insulin, glucagon and CD133, 9-23% single positive for glucagon, and 0-7% single positive for CD133, negative for insulin and glucagon, negative for insulin and CD133, and negative for insulin. Overall, the cultured cell population was determined to be 65% or more positive (triple positive) for insulin, CD133 and glucagon. (See FIG. 1) EXAMPLES

[0044] Populations of insulin- and glucagon-secreting pancreatic cells generated from non-insulin-producing pancreatic tissue obtained by needle biopsy from donors with type 1 diabetes secrete insulin in response to glucose stimulation in vitro.

[0045] To examine the glucose responsiveness of insulin- and glucagon-secreting cells expanded from T1D biopsy tissue (see Example 1), insulin- and glucagon-secreting cells were subjected to a glucose-stimulated insulin secretion assay. Approximately 1×10 6 Cells / well were plated in 6-well dishes and underwent two stimulation conditions to assess insulin secretion. They were incubated for 30 min with either (1) islet cell culture medium (see Table 2) or (2) islet cell culture medium supplemented with a higher glucose concentration (final concentration 16.7 mM, as a stimulus for insulin secretion). After incubation, the supernatants were stored at -20°C until undergoing a standard ELISA assay for insulin quantification. Cells cultured in islet cell culture medium supplemented with a higher glucose concentration were shown to secrete higher insulin amounts than cells treated with standard islet cell culture medium (unstimulated control). Insulin secretion after glucose stimulation was relatively increased compared to the unstimulated control, and more specifically, higher insulin amounts (95+ / -11 pMol / L) were seen in stimulated cultures compared to cells treated with standard islet cell culture medium (32+ / -7 pMol / L "unstimulated control"). Referring to FIG. 2, the stimulation index (an index of the ratio of insulin secretion induced by high glucose to basal secretion) of cells treated with islet cell culture medium supplemented with high glucose is shown compared to unstimulated controls. EXAMPLES

[0046] Characterization and mRNA analysis of insulin- and glucagon-secreting cells from T1D donor tissue. RNA sequencing was used to characterize several cell populations, including pancreatic tissue from deceased donors, insulin- and glucagon-secreting cells generated by the methods described herein, and de novo pseudo-islets from the same deceased donors. The properties of the various cell types were evaluated using the Illumina® NovaSeq™ platform. Markers evaluated were insulin, glucagon, PDX-1, SST, IIAP, Pax4, Pax-6, NKx2, Nkx6, NeuroD1, MafA, and MafB. We also found that cell compositions containing insulin- and glucagon-secreting cells generated from T1D-derived pancreatic tissue showed a significant decrease in markers of exocrine function (AMY and CTRC) expressed in the native pancreas. Concomitantly, expression of proliferation markers PCNA and CCND1 (cyclin family) increased, potentially indicating dedifferentiation into motile proliferating cells, consistent with observations of cell proliferation in vitro. Insulin- and glucagon-secreting cells generated from pancreatic tissue obtained by needle biopsy from T1D donors underwent morphological rearrangements and spontaneously generated de novo-pseudoislets when cultured for longer periods (~20 days or more). These islet-like structures were characterized by significantly increased expression of endocrine precursor and islet signature markers, including insulin, glucagon, PDX-1, SST, IIAP, Pax4, Pax-6, NKx2, Nkx6, NeuroD1, MafA, and MafB, and downregulated cell proliferation pathways. Furthermore, IGFBP1, a β-cell regeneration marker, was expressed at higher levels in the pseudoislets compared to cell compositions containing insulin- and glucagon-secreting cells. Meanwhile, stem cell markers LY6E and PROM1 showed higher expression in the cell compositions, suggesting that they had matured into more differentiated endocrine precursor cell populations committed to generating α, β, and δ cells.

[0047] Microarray mRNA profiles of these cells confirmed a gene profile compatible with a pancreatic endocrine islet cell population, and were found to express insulin and glucagon. Furthermore, these T1D-derived cell populations also expressed the pancreatic transcription factors PDX1, Nkx6, ngn3, NeuroD, and MafA and MafB, the islet neogenesis factor nestin, the glucose transporter Glut-2, the β-cell secretion product IAPP, and the islet δ-cell secretion somatostatin. These results suggested that the T1D-derived cell populations possessed all the factors required for islet neogenesis. Referring to Figure 3, we provide an overview of the up- and down-regulation of gene families in insulin- and glucagon-secreting cells from T1D pancreatic tissue. EXAMPLES

[0048] A method for increasing insulin secretion in vivo via transplantation of a cell composition comprising insulin- and glucagon-secreting cells into a host animal. To verify the efficacy of T1D-derived insulin- and glucagon-secreting cells as a treatment and transplantation method, four STZ-treated mice (NOD-SCID, 5–6 weeks old; Jackson Laboratory, Bar Harbor, ME) were inoculated with approximately 2.5 × 10 6 Biopsy-derived insulin- and glucagon-secreting cells from T1D mice (cells were counted using a Neubauer Chamber) were injected twice (total of 2 doses) at 1-week intervals. Follow-up was for 30 days. Blood was collected from the tail vein from day 14 after the first injection and at the end of follow-up. Serum was stored at -20°C until used to measure human insulin and human C-peptide concentrations by ELISA (Abcam and Alpco, respectively).

[0049] T1D derived insulin and glucagon secreting cells were generated using the methods described herein, for example, by culturing T1D pancreatic tissue in culture medium containing a polypeptide according to SEQ ID NO: 1 or 2 at concentrations ranging from 3 to 20 μg / ml. An example of a pancreatic islet cell culture medium is described as follows (and shown in Table 2 and Example 1): L-glutamine (2 mmol), ciprofloxacin (2 mg / L), amphotericin B (0.1 mg / L) penicillin (100,000 units / L) and streptomycin (100,000 micrograms / L), as well as a polypeptide according to SEQ ID NO: 1 or 2 (at 10 μg / ml) and CMRL supplemented with fetal calf serum (FCS) (10%), human serum (10%). Control culture medium is described in Table 3.

[0050] Before transplantation, cells were cultured for approximately 50-60 days. At the time of transplantation, cells were detached from the bottom of the plate / flask using trypsin (available from Gibco). After filtering through a 40 μm sterile mesh, single cells were counted by spinning (180g-300g for approximately 10 min) in phosphate buffer (calcium and magnesium free) or Hank's balanced salt solution (both available from Sigma Aldrich), and then aliquoting 1.25x10 cells into approximately 200 μl of sterile PBS (or HBSS). 6 The solution was resuspended in a volume of 100 μl / 100 μl and injected via the tail vein into anesthetized mice. Injections were given over 1 minute.

[0051] Human insulin was detected in all mice 14 and 30 days after the first injection (measured concentrations ranged from 12.5 to 33 pmol / L). Human C-peptide was measured on day 30 and confirmed the positive results (at levels down to 10 pmol).

[0052] As an alternative to intravenous injection, cells were suspended in PBS at a concentration of 20–50 μl and injected into the subcapsular space of the kidney (approximately 1 cm) using PE-50 tubing connected to a syringe. 2 The gene may also be inserted into the nucleoside 1 (occupying the region of the nucleoside 1 in the ribosome) (method described by Bertera et al., Journal of Transplantation).

[0053] Volume 2012 Article ID 856386, 9 pages doi:10.1155 / 2012 / 856386. Using this method, it is possible to administer a large number of cells at once (e.g., about 5-10 × 10 6 In contrast, intravenous injection of human cells yields 2.5 × 10 6 Higher doses may not be well tolerated.

[0054] The normal range of insulin in nondiabetic human serum is approximately 35.9–143.5 pmol / l, and considering limitations resulting from differences in the clearance of human insulin in mice compared with humans, the normal range is 3.0–25 × 10 6 It is reasonable to expect that administration of cells / kg body weight will provide a quantity of insulin that has the potential to impact glucose regulation. Dosing can be done once or multiple times, repeated every 3-6 months or even yearly, as needed. Dosage depends on many factors, including severity, sex, weight, and age. EXAMPLES

[0055] A method for increasing insulin secretion in vivo by transplanting a cell composition comprising insulin- and glucagon-secreting cells into a host animal. It has been determined that cell compositions comprising insulin- and glucagon-secreting cells grown from mouse islets using the islet cell culture media described herein can be safely injected and / or implanted via the kidney capsule into animals such as mice.

[0056] In one example, human insulin secretion was detected after implantation of insulin- and glucagon-secreting cells under the kidney capsule of mice for a period of at least 100 days (see FIG. 4).

[0057] The expanded insulin- and glucagon-secreting cells were harvested and then suspended in a solution such as Hank's Balanced Salt Solution (HBSS) or a solution containing phosphate-buffered saline (PBS) (available from Sigma Aldrich) to form a cell composition. In this example, a cell composition consisting of insulin- and glucagon-secreting cells suspended in Hank's Balanced Salt Solution was transferred under the kidney capsule of streptozotocin-diabetic (STZ) nude mice (5-6 weeks old; Jackson Laboratory, Bar Harbor, ME) using known methods such as the approach described in Bertera et al. 2012.

[0058] Briefly, mice were injected with streptozotocin (240 mg / kg IP) prior to transplantation and hyperglycemia (nonfasting blood glucose >350 mg / dl on two consecutive measurements) was confirmed.

[0059] On the day of transplantation, insulin- and glucagon-secreting cells are detached from the culture using trypsin or the like, centrifuged (300 g), and the detached cells are counted to prepare a cell composition. Approximately 4×10 6 The cells are suspended in a solution containing HBSS and loaded into a tube or catheter, such as a PE50 tube. The catheter or tube containing the cell composition is then placed under the kidney capsule of a fully anesthetized STZ mouse through a small incision in the left flank, followed by exposure of the kidney, to implant the cell composition into the STZ mouse.

[0060] At 14, 56, and 100 days after transplantation, blood was collected from the tail vein of STZ mice that received the cell composition, and plasma was separated and stored. Insulin levels were measured using an ELISA kit specific for human insulin (ALPCO Diagnostics, Salem, NH, USA). Referring to Figure 4, insulin levels in streptozotocin-induced diabetic mice treated, via transplantation, with a composition comprising insulin- and glucagon-secreting cells are shown. EXAMPLES

[0061] A method for treating hyperglycemia and diabetes by transplanting a cell composition comprising insulin- and glucagon-secreting cells into a host animal. Cell compositions containing insulin- and glucagon-secreting cells generated by treating non-type 1 diabetic pancreatic tissue (from humans and mice) with a stromal medium and a pancreatic islet cell culture medium containing a polypeptide according to SEQ ID NO: 1 or 2 have been shown to secrete insulin upon glucose stimulation at levels sufficient to lower blood glucose levels. Approximately 4×10 6 These cell compositions containing cells of not only secrete insulin and glucagon but also home to and engraft in the pancreas when injected intravenously. Cell compositions containing cells that secrete insulin and glucagon can be provided by cell transplantation for the treatment of pancreatic diseases, including diabetes and hyperglycemia.

[0062] Expression of the stem cell marker CD133 correlates with the long-term engraftment potential of the cells, and these cells have the innate ability to migrate and home to sites of injury. It has been determined that cell compositions as disclosed herein, when injected into STZ-diabetic mice, migrate to the pancreas and normalize hyperglycemia as shown by a corresponding reduction in blood glucose levels after treatment. For example, six (6)4 STZ-treated mice were injected with (approximately) 20×10 6 The recipient animals were injected with a cell composition comprising islet cells and treated with islet cell culture medium containing a polypeptide set forth in SEQ ID NO: 1 or 2. Results showed that by day 10, all mice showed reduced blood glucose levels, with 2 of 4 mice showing fasting blood glucose levels below 200 mg / dl by day 22 post-injection. All recipient animals had reduced blood glucose levels, with 2 animals maintaining blood glucose levels near 250 mg / dl and 1 animal having levels as low as 180 mg / dl on day 85 of the experiment. See FIG. 5.

[0063] Since cells from normal pancreatic tissue exhibit very similar characteristics to cells generated from T1D pancreatic samples, i.e., triple positive for CD133, glucagon, and insulin, it is reasonable to expect that a cell composition comprising insulin- and glucagon-secreting cells generated from a T1D pancreatic tissue sample treated with islet cell culture medium containing a polypeptide set forth in SEQ ID NO: 1 or 2 will result in a measurable reduction in blood glucose levels in vivo. Furthermore, transplantation of a cell composition comprising insulin- and glucagon-secreting cells has been shown to be non-tumorigenic, making it a desirable option for the treatment of pancreatic diseases by transplantation (either by injection, infusion, or engraftment). EXAMPLES

[0064] Culture, viability testing and cryopreservation methods for insulin- and glucagon-secreting pancreatic islet cells. In one embodiment, the pancreatic islet cell culture medium is CMRL-1066 (Mediatech, #99-663-CV; Transplantation Medium without Phenol Red (CMRL1066)): 10% heat inactivated fetal calf serum (Gibco, #16140071), 10% human serum (Gemini, #100512), L-glutamine (2 mM, Gibco, #25030081), ciprofloxacin (2 ml / L, B iworld, #403100313), amphotericin B (0.1 mg / L, Gibco, #15290026), penicillin-streptomycin (100,000 U / L-100,000 μg / L, Gibco, #10378016), and peptides according to sequence ID 1 or 2 (range of 3-20 μg / ml, e.g., 3 μg / ml, 5 μg / ml, or 10.0 μg / ml). In another embodiment, the cell culture medium for cryopreservation comprises cryoprotectant medium (Gibco #12648010). In another embodiment, viability testing was performed using a method comprising steps utilizing fluorescein diacetate (FDA) (Sigma Aldrich #F7378) and propidium iodide (PI) (Sigma Aldrich #P4170).

[0065] In one embodiment, human pancreatic tissue was collected by needle biopsy from a human donor suffering from type 1 diabetes or severe pancreatitis and cultured in vitro in pancreatic cell culture medium containing supplemented CMRL1066 on plates (or flasks) coated with 10% fetal bovine serum, 10% human serum, 2 mmol / L-glutamine, antibiotics, and peptides according to SEQ ID NO:1 or 2 at concentrations ranging from 3 μg / ml to 20 μg / ml, e.g., 100 μg / ml, and an attachment factor mixture (AFM) containing collagen type I and endothelial cell attachment factor (ECAF). Various ratios of ECAF to collagen were used, including a 50 / 50 ratio of collagen to ECAF. A thin layer of AFM (between 3 and 10 ml) was applied and allowed to set for 30 minutes, after which excess AFM was removed and the plate was allowed to dry in a hood for 45 minutes. The plate was then washed with phosphate buffered saline (PBS) to remove potential contaminants. The harvested pancreatic tissue was incubated on the AFM-treated flasks / plates in islet cell culture medium for 10-20 days until the cells began to mobilize and proliferate. The cultured cells were split when they reached approximately 70-80% culture confluence using standard sterile technique in a vertical laminar flow hood. For the splitting technique, the supernatant was removed from the culture plate (supernatant was saved). The plate was then washed with 2-5 ml of PBS (washing solution was saved). The cultured cells were detached using approximately 3-5 ml of trypsin (25% solution) by incubating in the presence of trypsin at 37°C for approximately 3-5 minutes until the cells detached. The plate was then washed a second time with PBS. The trypsinized cells, the saved PBS washing solution and the collected cell culture supernatant were then centrifuged at 1000 rpm for 7 minutes at 4°C. The resulting supernatant was decanted and the pellet was resuspended in 2 ml of PBS and centrifuged. The supernatant was then removed and the pellet was resuspended in islet cell culture medium containing sequence ID 1 or 2 at ~1000 cells / cm. 2 Cultures not used for replication were replated at a maximum cell density of 1x10 in cryopreservation medium. 6The cultured cells were resuspended at 1000 μg / ml. The cultured cells were centrifuged at 4° C. (1000 rpm, 7 min). The supernatant was aspirated and replaced with fresh cryopreservation medium, and the cells were transferred to a freezing container Mr. Frosty (Thermo-Fisher #5100-0036) overnight, and then transferred to and stored in vapor-phase liquid nitrogen.

[0066] Cells were subjected to a viability assay prior to cryopreservation: minimal viability acceptable for cryopreservation = 95% (hence 95% of all cells analyzed were stained with the viability fluorescent dye FDA).

[0067] Two aliquots of approximately 200 cells were transferred in a volume of approximately 50 μl into 400 μl of a solution containing FDA and PI (FDA at concentrations of 0.46 μM and 14.34 μM, respectively) in Eppendorf tubes. The cells were centrifuged (1000 rpm, 7 min), approximately 95% of the supernatant was aspirated, and the cells were transferred to the remaining liquid on a microscope slide using a micropipette. The cells were analyzed using a green / red filter set under a fluorescence microscope (Olympus model CKX3). Viable cells are stained green (FDA) and dead cells are stained red (PI). The proportion of viable cells was determined (expressed as a percentage) by two operators independently.

[0068] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be 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 in connection with any of the above aspects and various embodiments can be applied interchangeably between the different embodiments.

[0069] Aspects Aspect 1. A composition comprising a population of insulin- and glucagon-secreting cells generated from non-insulin-secreting pancreatic cells harvested via needle biopsy from a type 1 diabetic donor pancreas, a pancreatitis donor pancreas, or a combination thereof. Embodiment 2. The composition of embodiment 1, wherein at least about 50% of said insulin and glucagon secreting cell population expresses CD133, glucagon, and insulin. Embodiment 3. A composition according to embodiment 1, wherein about 50% to about 100% of the insulin and glucagon secreting cell population expresses CD133, glucagon, and insulin (specifications: e.g., about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%, etc., including all values ​​in between, specifying the percentage as a percentage of the total culture cell population). Aspect 4. The composition of aspect 1, wherein said insulin and glucagon secreting cell population is about 1.2×10 per kg of human recipient body weight. 6 Cells ~ approx. 25×10 6 The composition contains cells (specifications: e.g., about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, and all values ​​in between, specifying the mass (in kg) of a typical human recipient (e.g., pediatric (e.g.: 4-60 kg), adult (60-225 kg)). Embodiment 5. The composition of embodiment 1, wherein said non-insulin-secreting pancreatic cells harvested via needle biopsy are obtained from a donor pancreas of a type 1 diabetes patient. Embodiment 6. The composition of embodiment 1, wherein said non-insulin-secreting pancreatic cells harvested via needle biopsy are obtained from a pancreatitis donor pancreas. Embodiment 7. The composition of embodiment 1, wherein said non-insulin-secreting pancreatic cells are isogenic, allogenic, or a combination thereof. Embodiment 8. A method of treating a pancreatic disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of embodiment 1, wherein said pancreatic disorder comprises type 1 diabetes, pancreatitis, or a combination thereof. Embodiment 9. The method of embodiment 8, wherein said administering step comprises delivering the therapeutically effective amount of the composition of embodiment 1 to a target site in the subject via one or more of injection, infusion, ovarian or peritoneal pouch, surgical implantation, or packaging of the composition as part of a device. Embodiment 10. A method for preparing a composition comprising a population of insulin and glucagon secreting cells, comprising: 1. A method comprising the step of treating in vitro a non-insulin-secreting type 1 diabetic pancreatic cell population with a pancreatic islet cell culture medium having a base medium and an effective amount of a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or 2, SEQ ID NO:1, an active fragment of SEQ ID NO:1, SEQ ID NO:2, an active fragment of SEQ ID NO:2, or a combination thereof. Aspect 11. The method of aspect 10, wherein said treating step further comprises differentiating a non-insulin secreting type 1 diabetic pancreatic cell population and expanding said insulin and glucagon secreting cell population. Aspect 12. The method of aspect 10, wherein said treating step further comprises differentiating a non-insulin-secreting type 1 diabetic pancreatic cell population and expanding said insulin- and glucagon-secreting cell population, wherein at least about 50% of said insulin- and glucagon-secreting cell population express CD133, glucagon, and insulin. Aspect 13. The method of aspect 10, wherein the treating step further comprises differentiating a non-insulin-secreting type 1 diabetic pancreatic cell population and expanding the insulin- and glucagon-secreting cell population, wherein about 50% to about 100% of the insulin- and glucagon-secreting cell population express CD133, glucagon, and insulin. Aspect 14. The method of aspect 10, wherein said treating step further comprises differentiating a non-insulin secreting type 1 diabetic pancreatic cell population and expanding said insulin and glucagon secreting cell population, wherein said insulin and glucagon secreting cell population is about 3×10 per kg of human recipient body weight. 6 Cells ~ approx. 25×10 6 The method comprises the steps of: Embodiment 15. The method further comprising the step of extracting said non-insulin secreting type 1 diabetic pancreatic cell population from a donor. Embodiment 16. The method according to embodiment 10, further comprising the step of extracting the non-insulin secreting type 1 diabetic pancreatic cell population from the donor via needle biopsy. Example 17. The method of example 10, further comprising the step of extracting said population of non-insulin secreting type 1 diabetes pancreatic cells from an isogenic donor, an allogenic donor, or a combination thereof. Example 18. The method of example 10, wherein said pancreatic islet cell culture medium comprises the polypeptide in an amount ranging from about 3 μg / mL to about 20 μg / mL. Aspect 19. The method of aspect 10, wherein said treating step further comprises differentiating said non-insulin secreting type 1 diabetic pancreatic cell population and expanding said insulin and glucagon secreting cell population to obtain a therapeutically effective amount of said insulin and glucagon secreting cell population, and said method further comprises administering said therapeutically effective amount of said insulin and glucagon secreting cell population to a subject in need thereof. Aspect 20. A composition for use in treating type 1 diabetes, pancreatitis, or a combination thereof, comprising a population of insulin and glucagon secreting cells generated from non-insulin secreting pancreatic cells harvested via needle biopsy from a type 1 diabetic donor pancreas.

[0070] While the foregoing information emphasizes the aspects disclosed herein as illustrated and examples for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the subject matter claimed herein. It will be apparent to one of ordinary skill in the art that features described in connection with any of the above aspects and various embodiments may be applied interchangeably between the different embodiments.

[0071] The above aspects and embodiments are examples intended to illustrate various features of the subject matter claimed herein. All publications and patent applications disclosed in this specification are indicative of the level of ordinary skill in the art to which the subject matter of this disclosure and claims pertains.

[0072] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other moieties, additives, ingredients, or steps.

[0073] Throughout the description and claims of this specification, the singular encompasses the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the specification shall be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0074] It shall be understood that features, properties, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example may be applied to other aspects, embodiments, or examples described herein, unless incompatible therewith. All of the features disclosed herein (including the accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The subject matter claimed herein is not limited to the details of any of the foregoing embodiments. The subject matter claimed herein extends to any novel one, or any novel combination, of the features disclosed herein (including the accompanying claims, abstract, and drawings), or any novel one, or any novel combination of the steps of any method or process so disclosed.

[0075] All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by reference. Specific patent applications incorporated by reference include, for example, U.S. Patent Application No. 15 / 811,060, filed November 13, 2017 (and published as US2018 / 0133280A1); and International Patent Application No. PCT / US2019 / 038305, filed June 20, 2019 (and published as WO2020 / 005721A1). If there is a conflict between the terms and expressions disclosed herein and the terms and expressions incorporated herein within the scope of the terms and expressions, the information disclosed herein shall prevail.

Claims

1. A composition comprising a population of insulin- and glucagon-secreting cells generated from non-insulin-secreting pancreatic cells harvested from a type 1 diabetic donor pancreas, a pancreatitis donor pancreas, or a combination thereof.

2. The composition of claim 1, wherein at least about 50% of the population of insulin- and glucagon-secreting cells expresses CD133, glucagon, and insulin.

3. The composition of claim 1, wherein about 50% to about 100% of the population of insulin- and glucagon-secreting cells expresses CD133, glucagon, and insulin (specification: including all values in between, such as about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%, specifying the percentage as a total of the population of cells in the medium).

4. In the composition according to claim 1, the insulin and glucagon secreting cell population is about 1.2×10 ６ cells to about 25×10 ６ cells per kg of body weight of a human recipient (specification: including, for example, about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, and all values in between, and specifying the mass (in kg) of typical human recipients (e.g., pediatric (e.g.: 4 to 60 kg) and adult (60 to 225 kg))), composition.

5. The composition of claim 1, wherein the non-insulin-secreting pancreatic cells are harvested via tissue or needle biopsy and obtained from a donor pancreas of a type 1 diabetic patient.

6. The composition of claim 1, wherein the non-insulin-secreting pancreatic cells are harvested via tissue or needle biopsy and obtained from a pancreatitis donor pancreas.

7. The composition of claim 1, wherein the non-insulin-secreting pancreatic cells are isogenic, heterogenic, or a combination thereof.

8. A method of treating a pancreatic disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 1, wherein the pancreatic disorder includes type 1 diabetes, pancreatitis, or a combination thereof.

9. The method of claim 8, wherein the administering step comprises delivering the therapeutically effective amount of the composition of claim 1 to a target site of the subject via one or more of injection, infusion, ovarian, or peritoneal pouch, surgical transplantation, or via packaging of the composition as part of a device.

10. A method of preparing a composition comprising a population of insulin- and glucagon-secreting cells, comprising A method comprising the step of treating a non-insulin-secreting type 1 diabetic pancreatic cell population in vitro with a pancreatic cell culture medium having a basal medium and an effective amount of a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, SEQ ID NO: 1, an active fragment of SEQ ID NO: 1, SEQ ID NO: 2, an active fragment of SEQ ID NO: 2, or a combination thereof.

11. The method according to claim 10, wherein the step of treating further comprises the step of differentiating a non-insulin-secreting type 1 diabetic pancreatic cell population and the step of expanding the insulin- and glucagon-secreting cell population.

12. The method according to claim 10, wherein the step of treating further comprises the step of differentiating a non-insulin-secreting type 1 diabetic pancreatic cell population and the step of expanding the insulin- and glucagon-secreting cell population, and at least about 50% of the insulin- and glucagon-secreting cell population expresses CD133, glucagon, and insulin.

13. The method according to claim 10, wherein the step of treating further comprises the step of differentiating a non-insulin-secreting type 1 diabetic pancreatic cell population and the step of expanding the insulin- and glucagon-secreting cell population, and about 50% to about 100% of the insulin- and glucagon-secreting cell population expresses CD133, glucagon, and insulin.

14. In the method according to claim 10, the step of processing further includes a step of differentiating a non-insulin-secreting type 1 diabetic pancreatic cell population and a step of proliferating the insulin- and glucagon-secreting cell population, and the insulin- and glucagon-secreting cell population is about 3×10 ６ cells to about 25×10 ６ cells per kg of body weight of a human recipient.

15. The method further comprising the step of extracting the non-insulin-secreting type 1 diabetic pancreatic cell population from a donor.

16. The method according to claim 10, further comprising the step of extracting a non-insulin-secreting type 1 diabetic pancreatic cell population from a donor via a needle biopsy.

17. The method according to claim 10, further comprising the step of extracting the non-insulin-secreting type 1 diabetic pancreatic cell population from an isogenic donor, a heterogenic donor, or a combination thereof.

18. The method according to claim 10, wherein the pancreatic cell culture medium comprises an amount of polypeptide in the range of about 3 μg / mL to about 20 μg / mL.

19. The method according to claim 10, wherein the step of treating further comprises the step of differentiating the non-insulin secreting type 1 diabetic pancreatic cell population and expanding the insulin and glucagon secreting cell population to obtain a therapeutically effective amount of the insulin and glucagon secreting cell population, and the method further comprises the step of administering the therapeutically effective amount of the insulin and glucagon secreting cell population to a subject in need thereof.

20. A composition for use in the treatment of type 1 diabetes, pancreatitis, or a combination thereof, comprising an insulin and glucagon secreting cell population generated from non-insulin secreting pancreatic cells harvested from a type 1 diabetic donor pancreas.

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