CN117917961A

CN117917961A - Compositions and methods for proliferation of insulin and glucagon secreting cells from type 1 diabetic pancreatic tissue and therapeutic uses thereof

Info

Publication number: CN117917961A
Application number: CN202280061228.4A
Authority: CN
Inventors: 因告克·塔伊; 乔纳森·波莱特; 丽塔·博蒂诺
Original assignee: Imagination Pharmaceutical Co ltd
Current assignee: Imagination Pharmaceutical Co ltd
Priority date: 2021-09-22
Filing date: 2022-09-19
Publication date: 2024-04-23

Abstract

Disclosed herein are compositions and methods for producing compositions comprising cell-based therapeutic agents useful for treating pancreatic disorders, including type 1 diabetes.

Description

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

Citation of related applications

The present application claims priority from U.S. provisional patent application No. 63/247,252 filed on month 22 of 2021 and U.S. provisional patent application No. 63/337,137 filed on month 1 of 2022, the contents of which are incorporated herein by reference in their entirety.

Background

Cell-based therapies offer the hope of treating and altering the course of pancreatic diseases such as type 1 diabetes (T1D), which cannot be adequately addressed by existing therapies, however cell-based therapies present a number of problems, mainly related to safety and efficacy and scalability of preparation. Many of the problems associated with cell-based therapies are disclosed in Bashor, c.j., ENGINEERING THE next generation of cell-based therapeutics, nat Rev Drug Discov (2022) (and available on-line in https:// doi.org/10.1038/s 41573-022-00476-6).

Disclosure of Invention

Disclosed herein are compositions comprising cell-based therapeutic agents for treating pancreatic disorders (including type 1 diabetes) and methods of producing the compositions. In one embodiment, the compositions disclosed herein comprise a population of insulin-and glucagon-secreting cells produced by non-insulin-secreting pancreatic cells harvested from a type 1 diabetic donor pancreas via needle biopsy. In another embodiment, the compositions disclosed herein comprise a population of insulin and glucagon secreting cells produced by pancreatic cells harvested from a patient or donor suffering from chronic pancreatitis via needle biopsy. In one embodiment, non-insulin secreting type 1 diabetic pancreatic cells are treated in vitro with a pancreatic 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, wherein the treatment causes the treated cells to differentiate and proliferate into a population of pancreatic islet progenitor cells that secrete both insulin and glucagon in response to the stimulus and are CD133 positive. The resulting insulin and glucagon secreting progenitor cells can be propagated to the desired cell count for subsequent use in transplantation or injection, and as cell-based therapeutics for type 1 diabetes or chronic pancreatitis. The cell composition comprising an effective amount of the insulin-and glucagon-secreting progenitor cell population may be administered to a subject by infusion, injection, transplantation, portal intravenous delivery (intra portal delivery), or by other suitable delivery means, such as using a medical device, as a method for restoring insulin and glucagon secretion in response to a stimulus (stimulus, stimuli).

The compositions and methods disclosed herein are of interest for producing large quantities of insulin and glucagon secreting pancreatic cells that are useful in cell-based therapies and cell transplantation, i.e., autologous or allogenic transplantation for the treatment of type 1 diabetes or chronic pancreatitis.

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 a population of pancreatic cells that secrete insulin and glucagon, wherein the population of pancreatic cells that secrete insulin and glucagon is produced by treating the pancreatic cells with an islet cell culture medium comprising a basal medium and a peptide comprising an amino acid sequence according to SEQ ID 1 or 2, the pancreatic cells collected from diseased pancreatic tissue (e.g., from a type 1 diabetic subject or a subject having chronic pancreatitis), for example, via needle biopsy. When administered to a subject in need thereof, the composition delivers healthy pancreatic progenitor cells to a target site of the subject, wherein the healthy pancreatic progenitor cells are capable of producing insulin and glucagon in response to the stimulus.

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 therapeutics to supplement the loss of insulin production or replace insulin production in patients with type 1 diabetes or other diseases characterized by severe insulin deficiency (e.g., after complete or partial pancreatectomy, with and without autologous or allogeneic islet transplantation).

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

In another embodiment, an islet cell culture medium for stimulating the growth, proliferation and differentiation of insulin and glucagon secreting cells from pancreatic cells derived from type 1 diabetic pancreatic tissue comprises a basal medium and an effective amount of a polypeptide, wherein the polypeptide comprises an amino acid sequence according to one or more of SEQ ID nos. 1-2 (listed in table 1), or an active fragment thereof. In one embodiment, the polypeptide comprises an amino acid sequence having at least 50% sequence identity with 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; 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.

In yet another embodiment, a cell composition comprises a population of insulin-and glucagon-secreting cells produced by treating isolated type 1 diabetic pancreatic tissue with an islet cell culture medium comprising basal 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 cell to glucose; wherein the cell composition comprises a population of cells capable of secreting insulin and glucagon in response to an appropriate stimulus.

In another embodiment, a method of preparing a cell composition comprises in vitro administering an islet cell culture medium comprising a basal medium and an effective amount of a polypeptide according to SEQ ID No.1 or 2, or an active fragment thereof, to human pancreatic tissue harvested from a type 1 diabetic patient; incubating cells in islet cell medium; screening the incubated cells for one or more cell markers selective for CD133 and insulin; and collecting cells identified by screening from the cultured cell population for CD133 and insulin positivity; the cultured cells continue to grow until the desired number of cells proliferate.

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 restoring insulin production and secretion. The cell composition may be packaged as a delivery solution or in a delivery vehicle and administered by implantation, injection or infusion, the administration being systemic, local or localized to the target site.

In yet another embodiment, a method of treating a pancreatic disorder in a mammal (wherein the pancreatic disorder is characterized by an insufficient production of insulin) comprises: culturing in vitro a population of insulin and glucagon secreting cells from pancreatic tissue harvested from a type 1 diabetes donor pancreas in an islet cell culture medium comprising a basal medium and an effective amount of a polypeptide according to SEQ ID No.1 or 2, or an active fragment thereof, thereby producing a CD133 positive population of insulin and glucagon secreting cells; further comprising isolating and expanding the population to produce a population of cells that secrete predominantly (at least 60% or more) insulin and glucagon; and further comprising collecting insulin and glucagon-secreting cells and suspending the collected cells in a physiological buffer such as Phosphate Buffered Saline (PBS) or Hanks Balanced Salt Solution (HBSS), and implanting or injecting into the mammal a cell composition comprising insulin and glucagon-secreting cells in suspension with the physiological buffer. In one embodiment, the composition may be delivered as an aqueous solution, suspension, capsule, microcapsule, and/or encapsulated or semi-solid formulation; wherein the composition may be delivered to the mammal via one or more of injection, infusion, omentum or peritoneal bags, surgical implantation, or via packaging of the composition as part of a device to a target site of the mammal.

In another embodiment, the cell composition comprises a population of cells that secrete insulin and glucagon, further comprising one or more of a buffer, a pharmaceutically acceptable carrier, a pharmaceutically acceptable additive, an antibiotic, or other agent.

Drawings

The compositions and methods disclosed herein are further described by the accompanying figures. The term "IPC" is used in the figures to refer to Insulin Producing Cells (IPC) as described and claimed herein.

Figure 1 shows that greater than 50% of T1D-secreting cells that proliferate according to the methods herein have triple positives for CD133, insulin, and glucagon.

Fig. 2 shows that T1D pancreatic tissue cultured with islet cell culture medium comprising a peptide according to SEQ ID No.1 or 2 produces cells that secrete insulin in response to glucose stimulation (as indicated by the stimulation index), which is the ratio between insulin secretion under high glucose conditions relative to basal release under non-stimulated conditions. A value higher than 2 indicates glucose responsiveness in the cell. Sample 1 is a population of cells proliferated from normal pancreatic tissue according to the methods herein; samples 2-4 are cell populations proliferated from T1D pancreatic tissue according to the methods herein.

Figure 3 shows down-and up-regulation of genes (families) associated with pancreatic function in cell preparations derived from single T1D biopsies compared to native pancreatic tissue. Each gene family includes 5-13 genes. As shown, the cell populations comprising insulin and glucagon secreting cells produced according to the methods herein exhibit up-regulation of the gene families necessary for mature islet cells, beta cell maturation, GSIS, insulin particles, and cell cycle.

Fig. 4 shows serum insulin levels after transplantation of a cell composition comprising insulin and glucagon secreting cells propagated according to the methods herein in streptozotocin (STZ, a β -cell specific toxin that induces irreversible damage to islets of langerhans and induces diabetes) treated mice. The cell composition is shown to promote in vivo secretion of human insulin, which is present in serum of STZ mice treated with the cell composition disclosed herein for up to 100 days.

Fig. 5 shows that insulin and glucagon secreting cells produced from type 1 diabetes (T1D) cells proliferated according to the methods herein can normalize blood glucose levels following injection in a STZ diabetes mouse model. M1-4 refers to STZ mouse sample number, i.e., mouse-1, mouse-2, mouse-3, mouse-4.

Detailed Description

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

As used herein, "SEQ ID NO 1 or 2" refers to a protein, polypeptide, peptide fragment or analog thereof, and includes any altered sequence thereto, having an amino acid sequence with at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence according to SEQ ID NO 1 or 2 (see table 1). Peptide fragments or analogs thereof are also contemplated and include any altered sequences thereto, having an amino acid sequence with at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequences according to SEQ ID NOs 1 and 2. Treatment of cells (keratinocytes, intestinal cells, islet cells, endothelial cells and lung cells) with a polypeptide according to SEQ ID NO 1 or SEQ ID NO 2 added to a cell culture medium has been demonstrated to cause stimulation and increased cell growth by in vitro studies with polypeptides according to SEQ ID NO 1 and SEQ ID NO 2, yielding living progenitor cells as measured by the percentage of CD133 positive cells in culture and the MTT cell proliferation assay (SIGMA ALDRICH CELL Proliferation Kit). These progenitor cells can regenerate and proliferate to billions.

As used herein, the term "insulin and glucagon secreting cells" or "insulin and glucagon secreting islet cells" or "insulin and glucagon secreting progenitor cells" are used interchangeably herein to refer to a cell composition comprising insulin and glucagon secreting cells and/or cell populations that are produced from non-insulin secreting T1D pancreatic cells according to the methods described herein and are positive for the cellular markers CD133, insulin, glucagon, and produce insulin and glucagon in response to a stimulus, and are further characterized by the cellular markers PDX-1, SST, IIAP, pax4, pax6, nkx2, nkx6, neuroD1, mafA, mafB.

The term "proliferation" refers to an increase in the number of cells present in a culture due to cell division.

As used herein, "cultured," "cultured" or "cultured with" refers to the removal or isolation of cells from an environment (e.g., in a host mammal) and their subsequent growth in an advantageous in vitro artificial environment. "cultured cells" is intended to include subcultured (i.e., passaged) by transferring the cells into a new vessel with fresh growth medium to provide more room for continued growth, differentiation, and/or proliferation. References to "pancreatic cells" include those cells typically found in the pancreas of a mammal, and include islet cells, e.g., glucagon-synthesizing alpha cells, insulin-producing beta cells, and any combination thereof.

The term "target site" as used herein refers to a region in a recipient host (mammal, preferably human) that requires treatment or supplementation. The target site may be a single region within a particular organ or may be multiple regions in the host. In some embodiments, the supplementation or replacement produces the same physiological response as normal tissue (e.g., pancreatic tissue), whether or not the pancreas is targeted.

As used herein, the terms "treat," "treating" or "therapeutic treatment" and other grammatical equivalents as used herein include alleviating, slowing or ameliorating the symptoms of a disease or condition, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., preventing the development of a disease or condition, alleviating a disease or condition, causing the disease or condition to resolve, alleviating the symptoms of a disease or condition, or stopping the symptoms of a disease or condition, and preventing. These terms further include achieving a therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit refers to eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, but the patient is still likely to suffer from the underlying disorder.

As used herein, "effective amount" refers to an amount sufficient to achieve the effect. A therapeutically effective amount for treating a condition is an amount that achieves a clinically relevant endpoint in a patient or patient population. As a non-limiting example, the composition comprising insulin and glucagon secreting cells is administered in an amount of about 1.2 to about 2.5x10 ⁶ cells/kg, or greater than 200 x 10 ⁶ cells, to produce sufficient insulin to cause a decrease in blood glucose level to about 100 to 125mg/dl (5.6 to 6.9 mmol/L), or to below 250mg/dl. Other ranges include about 3 x 10 ⁶ cells to about 25 x 10 ⁶ cells/kg body weight; or about 5 x 10 ⁶ to about 10 x 10 ⁶ million cells/kg. The appropriate dosage of the composition may depend on the route of administration, such as injection or infusion or transplantation, and may depend on the subject to be treated and the severity of the condition to be treated. Suitable and exemplary dosage ranges for administration of the compositions disclosed herein to adults can be predicted using a scaling method, such as differential scaling (allometric scaling). Dose escalation is an empirical method that is well characterized and understood in the art. This approach assumes that there are some unique features between species regarding anatomical, physiological and biochemical processes, and that the possible differences in pharmacokinetic/physiological times are themselves explained by magnification. As an example, not intended to be limiting, based on literature, human pancreas has between 6 x 10 ⁵ to about 2x 10 ⁶ islets; thus, there are about 600×10 ⁶ islet cells in a normal human pancreas, half of which are beta cells. Given that 30% of the islet mass is sufficient to maintain normal blood glucose, a 1.2X10 ⁶ dose of the insulin and glucagon secreting cells disclosed herein is expected to be sufficient to replace the insulin producing capacity of the non-diseased human pancreas.

As used herein, the term "sequence identity" refers to identity between two or more amino acid sequences that is expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percentage, the higher the sequence identity. Percent identity was calculated over the entire length of the sequence. Homologs or orthologs of amino acid sequences have a relatively high degree of sequence identity when aligned using standard methods. Such homology is more pronounced when the orthologous protein is derived from more closely related species (e.g., human and mouse sequences) than from more closely related species (e.g., human and caenorhabditis elegans (c. Elegans) sequences). Sequence alignment methods for comparison are well known in the art. Various procedures 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, J.mol. Biol.215:403-10,1990 provides detailed considerations for sequence alignment methods and homology calculations. The sequence identity level can be determined using NCBI Basic Local ALIGNMENT SEARCH Tool (BLAST) (Altschul et al, J.mol. Biol.215:403-10, 1990) which is available from a variety of sources, including the national center for biological information (NCBI, national Library of Medicine, building 38A,Room 8N805,Bethesda,Md.20894,US) and on the Internet.

It should be understood that a numerical value may be associated with a certain amount of experimental error. Thus, reference to the qualifier "about" (or "approximately") prior to a numerical error is intended to reflect an experimental error that may be associated with the numerical value. To the extent that the experimentally obtained value is not previously stated as "about" (or "approximately"), it is not intended that the value be associated with a certain amount of experimental error.

Representative cultures of insulin and glucagon secreting cells characterized herein have been deposited at ATCC [ accession number ___ ] at 9, month 7 of 2022 under the terms of the Budapest treaty. Cultured cells, proliferating cells, isolated cells, etc. may be protected from external mutagenic stimuli, such as UV radiation.

Using the methods disclosed herein, it has been determined that 30 days after isolation of T1D pancreatic tissue, a single pancreas can produce 770 billions of insulin and glucagon secreting (islet) cells; by day 60, 2 trillion cells are sufficient for infusion of 100-150 patients in need of treatment (depending on the severity of the disease or the dose used), or for storage (freezing/storing) of the cells for future expansion and reinfusion.

The amino acid residues of the active agent may be post-translationally modified or bound to other functional or nonfunctional molecular groups. For example, see Guo et al mol. Biosystem.7 (7): 2286-2295,2011, in which antagonistic citrullination and methylation of human ribosomal protein S2 (e.g., SEQ ID NO. 1) are generally described. Naturally, such modified amino acid residues are included in the amino acid sequences described herein and are included within the scope of the active agent.

The polypeptides and/or polypeptide fragments according to, for example, SEQ ID NOs 1 and 2 may be produced under protein production conditions known in the art, for example in bacteria, yeast, or by synthetic means, or as described in U.S. patent application Ser. No. 15/811,060.

In one embodiment, the cell composition may be packaged as a delivery solution, or in a medical device comprising a delivery vehicle, and may be administered by implantation, injection, or infusion, the administration being parenteral, systemic, local, or localized to a target site. In one embodiment, insulin and glucagon secreting islet cells produced in vitro are encapsulated and implanted into a mammal have been previously characterized in the art (see, e.g., altman, et al, 1984,Trans.Am.Soc.Art.Organs 30:382-386, and U.S. Pat. No.6,703,017b1, incorporated herein by reference) -and will be suitable for insulin and glucagon secreting cells produced according to the methods disclosed herein. Preferably, the encapsulant is hypoallergenic, is easily and stably located in the target tissue, and provides additional protection to the implanted cell composition to protect and prevent the implanted cells from being damaged.

The appropriate implant dose in humans can be determined from existing information about ex vivo islet transplantation in humans, further in vitro and animal experiments, and from human clinical trials. From data relating to ex vivo islet transplantation in humans, it is expected that about 8,000-12,000 islets per patient kg may be required. It is believed that long term survival of the implant after implantation, less than the number of naturally occurring islets (about 200 tens of thousands in a normal adult pancreas) or possibly even less than the amount used in an ex vivo islet implantation may be necessary.

In one embodiment, the cell composition has therapeutic benefit in a mammal for treating a pancreatic disorder, wherein the pancreatic disorder is hyperglycemia, type 1 diabetes, or chronic pancreatitis, comprising: administering a therapeutically effective amount of a population of cells that secrete insulin and glucagon, thereby providing treatment for a pancreatic disorder.

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, capsule, microcapsule, and/or encapsulated or semi-solid formulation; wherein the composition may be delivered to a patient in need thereof via one or more of injection, infusion, omentum or peritoneal bags, surgical implantation, or via delivery to a target site of a mammal by packaging the composition as part of a device.

In one embodiment, the composition comprises a population of insulin-and glucagon-secreting progenitor cells, further comprising one or more pharmaceutically acceptable excipients, and/or one or more pharmaceutically acceptable additives and/or one or more agents. Suitable excipients and additives include, but are not limited to, buffers such as PBS or HBSS, amino acids, stabilizers or compatibilizers (bulking agents, bulking agent), surfactants, antimicrobial/preservative agents, antifungal agents, metal ions/chelating agents, polymers, polyanions, salts, sugars, cyclodextrin-based excipients, lyoprotectants, solubilizing agents, antioxidants, complexing agents, anti-tacking agents, dispersing agents, serum additives.

In another embodiment, the present disclosure provides a method for treating a mammal, preferably a human, suffering from or at risk of developing type 1 diabetes or severe pancreatitis, the method comprising: removing pancreatic tissue from a mammal; culturing the resected pancreatic tissue in vitro to proliferate a population of insulin-and glucagon-secreting islet cells; and implanting, transplanting, infusing, injecting or otherwise inserting the insulin and glucagon secreting islet cell population into a mammal, alone or in combination with a medical or delivery device.

Examples

The following examples are provided by way of illustration, and not by way of limitation, of the subject matter claimed herein.

The cell cultures used in these examples were incubated at 37 ℃ under standard CO (5%) conditions; culturing (plating, separation of cells) was performed in a vertical laminar flow hood (VERTICAL LAMINAR flow hood) using standard aseptic techniques and conditions. Cells were cultured (including controls) in the islet cell media described in tables 2 and 3, unless otherwise indicated. When they reach about 70-80% confluence in culture, the cells are isolated.

In one embodiment, the separation technique (SPLITTING TECHNIQUE) involves removing the supernatant (preserving the supernatant) from the culture plate. The plate was then washed with 2-5ml PBS (preserve wash). Cells were dissociated (detach) using about 3-5ml trypsin (as available from Sigma-Aldrich) by incubating the cells in the presence of trypsin for about 3-5 minutes at 37 ℃ until the cells dissociated. The plates were then washed a second time with PBS. The trypsin-treated cells were then centrifuged at 300g for 7 minutes at 4 ℃ along with the stored PBS wash and the collected cell culture supernatant.

In one embodiment, the resulting supernatant is decanted, the pellet (pellet) is resuspended in 2ml PBS and centrifuged again. The supernatant was then removed and the pellet was resuspended in medium containing either SEQ ID 1 or 2 and re-plated at a cell density of about 1000 cells/cm ². While embodiments may refer to a cell culture plate, it should be understood that a cell culture flask is an acceptable substitute for a plate.

Example 1

A method of producing a population of insulin-and glucagon-secreting pancreatic cells from non-insulin-producing pancreatic tissue harvested from a type 1 diabetes donor by needle biopsy.

Human pancreatic tissue was collected from a type 1 diabetes (T1D) donor patient (58 year old female; 53 year old diabetes) via needle biopsy. Biopsy samples of 1 x 1mm ³ were obtained from donor pancreas (from organ restoration and education centers) and stored on ice in commercially available solutions (sold under the names Viaspan, belzerUW, bel-Gen or StoreProtec).

The harvested T1D tissue is then cultured in islet cell culture medium (see table 2) comprising: CMRL (such as phenol red-free Mediatech #99-663-CV transplanting medium (CMRL 1066)) 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), and polypeptides according to SEQ ID NO.1 or 2 (in the range of 3-20 micrograms/ml, and in particular 10 micrograms/ml), together with Fetal Calf Serum (FCS) (10%) and human serum (10%). The control medium contained: CMRL medium 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), as well as Fetal Calf Serum (FCS) (10%) and human serum (10%) (without addition of polypeptides according to SEQ ID NO.1 or 2). Standard tissue/cell culture conditions (37 ℃,5% co ₂) were used.

Tissues were cultured on plates or in flasks 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 may be used, including but not limited to a 50/50 ratio of collagen to ECAF. Briefly, plates (or bottles) were prepared by applying a thin layer of AFM (between 3-10 ml) to the plates, and after 30 minutes of standing, the excess AFM was removed. The panels were allowed to dry in a fume hood for 45 minutes. Prior to use, the plates were washed with PBS to remove any potential contaminants. The harvested tissue is incubated on AFM treated plates in islet cell culture medium supplemented with a polypeptide according to SEQ ID No.1 or 2 until the cells start to move and proliferate (about 10 to 20 days).

After 12-15 days of culture, cells from the biopsy move and begin to adhere to the dish and proliferate. During the following 4-6 weeks, adherent cells continued to proliferate, began to pool and continued to multiply, doubling every 3 days. The biopsy-derived cells in culture exhibited a morphology similar to that of the cell composition comprising insulin-secreting cells produced from non-diabetic donors (previously characterized in U.S. 2021/0205371 published at 7/8 of 2021). Similarly, cells form islet-like cell clusters that are consistent in size with Langerhans islets and exhibit insulin secretion in response to stimulation with glucose.

The expression of CD133 from the resulting cell cultures of T1D pancreatic biopsy tissue was determined by Fluorescence Activated Cell Sorting (FACS) using a flow cytometer (Becton Dickinson FACS Aria cell sorter) and intracellular insulin and glucagon expression. Cultured cells were first labeled for CD133 expression and then fixed and permeabilized with FOXP3 fixation/permeabilization buffer according to the manufacturer's instructions and stained with a coupled fluorometric antibody for glucagon and intracellular insulin, respectively. FACS analysis was performed after FOXP3 fixation permeation and staining with conjugated fluorometric antibodies. Cells derived from T1D pancreatic biopsies cultured in islet cell culture medium comprising the peptide according to SEQ ID No.1 or 2 were found to be positive for CD133, glucagon and insulin (herein referred to as "triple positive"), in particular 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, and negative for insulin and glucagon; negative for insulin and CD 133; and negative for insulin. In summary, it was determined that greater than 65% of the cultured cell population was positive for insulin, CD133 and glucagon (triple positive). (see FIG. 1)

Example 2

Insulin and glucagon secreting pancreatic cell populations are generated from non-insulin producing pancreatic tissue harvested by needle biopsy from a type 1 diabetes donor that secretes insulin in response to an in vitro glucose stimulus.

To test the glucose responsiveness of insulin and glucagon secreting cells proliferated from T1D biopsy tissue (see example 1), glucose stimulated insulin secretion assays were performed on insulin and glucagon secreting cells. Approximately 1×10 ⁶ cells/well were plated in 6-well dishes and subjected to 2 stimulation conditions to assess insulin secretion. The cells were incubated for 30 minutes with either: (1) islet cell culture medium (see table 2); or (2) islet cell culture medium supplemented with a higher glucose concentration (final concentration 16.7mM as a stimulator of insulin secretion). After incubation, the supernatant was stored at-20 ℃ until standard ELISA assays for insulin quantification were performed. Cells cultured in islet cell culture medium supplemented with higher glucose concentrations showed higher secretion of insulin than those treated with standard islet cell culture medium (unstimulated control). There was a relative increase in insulin secretion following glucose stimulation compared to the unstimulated control, and more specifically, higher amounts of insulin (95+/-11 pMol/L) were observed in the stimulated cultures compared to those cells treated with standard islet cell medium (32+/-7 pMol/L "unstimulated control"). Referring to fig. 2, the stimulation index (a measure of the ratio between insulin secretion with high glucose relative to basal release) of cells treated with islet cell medium supplemented with high glucose compared to unstimulated controls is shown.

Example 3

Characterization and mRNA analysis of insulin and glucagon secreting cells derived from T1D donor tissue.

Using RNA sequencing methods, characteristics of various cell populations were determined, including pancreatic tissue from a dead donor, insulin and glucagon secreting cells produced by the methods disclosed herein, and re-pseudo islets (denovo-pseudo-islets) from the same dead donor. UsingThe NovaSeq ^TM platform evaluates characteristics of different cell types. The markers evaluated were: insulin, glucagon, PDX-1, SST, IIAP, pax4, pax-6, NKx, nkx6, neuroD1, mafA and MafB. Also identified are significant reductions in the markers (AMY and CTRC) that exhibit exocrine function in cell compositions comprising insulin-and glucagon-secreting cells produced from T1D-derived pancreatic tissue, which are expressed in the native pancreas. Meanwhile, in the cell composition, the increased expression of proliferation markers PCNA and CCND1 (cyclin family), potentially indicative of dedifferentiation into actively proliferating cells, is consistent with the observation of cell expansion in vitro. After a longer incubation (about 20 days or longer), insulin and glucagon secreting cells produced by needle biopsy from pancreatic tissue harvested from T1D donors undergo morphological rearrangements and spontaneously produce re-pseudo islets. These islet-like structures are characterized by significantly increased expression of endocrine progenitor cells and islet tag markers (signaling markers) including insulin, glucagon, PDX-1, SST, IIAP, pax4, pax-6, NKx2, nkx6, neuroD1, mafA, and MafB, while exhibiting down regulation of the cell proliferation pathway. Furthermore, IGFBP1, a marker of β -cell regeneration, was found to be expressed at higher levels in pseudo islets when compared to a cell composition comprising insulin and glucagon secreting cells, whereas stem cell markers LY6E and PROM1 were expressed higher in the cell composition, indicating maturation into a more differentiated population of endocrine progenitor cells dedicated to the production of α, β and δ cells.

The microarray mRNA profile of the cells confirmed a genetic profile compatible with the pancreatic endocrine islet cell population with insulin and glucagon expression. In addition, these T1D-derived cell populations also express the pancreatic transcription factors PDX1, nkx6, ngn3, neuroD, and MafA and MafB, as well as the islet neogenesis factor nestin, the glucose transporter Glut-2, the secretion product IAPP of β -cells, and somatostatin secreted by islet δ -cells. These findings indicate that the T1D-derived cell population carries all factors necessary for islet neogenesis. Referring to fig. 3, an overview of up-and down-regulation of the gene families in insulin and glucagon secreting cells derived from T1D pancreatic tissue is shown.

Example 4

A method of increasing insulin secretion in vivo by transplanting into a host animal a cellular composition comprising insulin-and glucagon-secreting cells.

To test the effectiveness of T1D-derived insulin and glucagon secreting cells as a therapeutic agent and method for transplantation, four STZ-treated mice (NOD-SCID, 5-6 weeks old; jackson Laboratory, bar Harbor, ME) were injected with approximately 2.5 x10 ⁶ T1D-derived biopsy-derived insulin and glucagon secreting cells (counted twice a week using Neubauer Chamber) at intervals of 2 doses total. Tracking (follow up) for 30 days. Blood samples were obtained via the tail vein 14 days after the first dose, and at the end of the follow-up. Serum was obtained and stored at-20 ℃ until used to measure human insulin and human C peptide concentrations by ELISA (Abcam and Alpco, respectively).

Insulin and glucagon secreting cells derived from T1D are produced using the methods described herein, for example by culturing T1D pancreatic tissue in a medium comprising a polypeptide according to SEQ ID No.1 or 2 in a concentration in the range of 3-20 μg/ml. An example of islet cell culture medium is described below (and shown in table 2 and example 1): CMRL, 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), and polypeptides according to SEQ ID NO.1 or 2 (10 μg/ml), as well as Fetal Calf Serum (FCS) (10%) and human serum (10%). The control medium is described in table 3.

Cells were cultured for about 50-60 days prior to transplantation. At the time of transplantation, cells were dissociated from the bottom of the plate/flask using trypsin (available from Gibco). After filtration through a 40 μm sterile screen, the single cells were washed in phosphate buffered saline (without calcium and magnesium), hanks balanced salt solution (both available from SIGMA ALDRICH), spun (180 g-300g for about 10 minutes), counted and resuspended in about 200 μl of sterile PBS (or HBSS) at a dose of 1.25×10 ⁶/100 μl, and injected into anesthetized mice via the tail vein. Injection was performed over the course of one minute.

Human insulin was detected in all mice on days 14 and 30 (measured concentrations ranging from 12.5 to 33 pmol/L) after the first injection. When measured on day 30 (at levels up to 10 pmol), positive results for human C peptide were confirmed.

As an alternative to intravenous injection, the cells may be resuspended in PBS in a more concentrated volume of 20-50 μl and inserted into the subcapsular space (sub capsular space) of the kidney (occupying an area of about 1cm ²) by using a PE-50 tube connected to a syringe (method described by Bertera et al,Journal of Transplantation Volume 2012,Article ID 856386,9pages doi:10.1155/2012/856386). Using this method, a greater dose of cells (e.g., about 5-10 x 10 ⁶ cells) can be administered at a time compared to intravenous injection of human cells (where a dose of greater than 2.5 x 10 ⁶ cells may not be well tolerated).

Considering that the normal range of insulin in human serum of non-diabetic individuals is about 35.9 to 143.5pmol/l and considering the limitation of differences in clearance of human insulin compared to humans from mice, it is reasonably expected that doses of 3.0 to 25 x 10 ⁶ cells/kg of human receptor body weight will supply insulin in amounts having the potential to affect glucose regulation. The administration may be repeated one or more times every 3 to 6 months or annually, as desired. The dosage will depend on many factors such as the severity of the disease, sex, weight and age.

Example 5

It has been determined that using the islet cell culture media described herein, a cell composition comprising insulin and glucagon secreting cells proliferated from murine islets can be safely injected and/or transplanted into an animal (e.g., a mouse) via the kidney capsule (kidney capsule).

In one embodiment, human insulin secretion is detected for a period of at least 100 days after implantation of insulin and glucagon secreting cells under the kidney capsule of the mouse (see fig. 4).

The proliferated insulin and glucagon secreting cells are collected and then suspended in a solution, such as a solution comprising Hanks Balanced Salt Solution (HBSS) or Phosphate Buffered Saline (PBS) (available from SIGMA ALDRICH), to form a cellular composition. In this example, a cell composition comprising insulin and glucagon secreting cells suspended in Hanks balanced salt solution was transplanted under the kidney capsule of a streptozotocin-diabetic (STZ) nude mouse (5-6 weeks old; jackson Laboratory, bar Harbor, ME) using known methods, such as the method described in Bertera et al 2012.

Briefly, mice were injected with streptozotocin (240 mg/kg IP) and hyperglycemia was confirmed (non-fasting blood glucose levels >350mg/dl on 2 consecutive readings) prior to transplantation.

On the day of transplantation, cell compositions are prepared by dissociating insulin and glucagon secreting cells from the culture, for example, with trypsin; the cells were centrifuged (at 300 g) and dissociated cells were counted. About 4×10 ⁶ cells were suspended in a solution containing HBSS; and loaded into a tube or catheter (e.g., a PE50 tube). The cell composition was then transplanted into STZ mice by placing a catheter or tube containing the cell composition under the kidney capsule of a fully anesthetized STZ mouse, through a small incision in the left flank and subsequent exposure of the kidney.

On days 14, 56 and 100 post-transplantation, blood was obtained from the tail vein of STZ mice receiving the cell composition, and plasma was isolated and stored. Insulin levels were measured using an ELISA kit (ALPCO Diagnostics, salem, NH, USA) specific for human insulin. Referring to fig. 4, insulin levels in streptozotocin-diabetic mice treated via transplantation with a composition comprising insulin-and glucagon-secreting cells are shown.

Example 6

Methods for treating hyperglycemia and diabetes by transplanting into a host animal a cellular composition comprising insulin and glucagon secreting cells.

Cell compositions comprising insulin and glucagon secreting cells produced by treating non-type 1 diabetic pancreatic tissue (human and murine) with islet cell culture medium comprising basal medium and a polypeptide according to SQ ID NO:1 or 2 have been shown to secrete insulin at levels sufficient to reduce blood glucose following glucose stimulation. When injected intravenously, these cell compositions, which contain approximately 4×10 ⁶ cells, not only secrete insulin and glucagon, but also pool (home) and implant (engraft) into the pancreas. Cell compositions comprising insulin and glucagon secreting cells can be delivered via cell transplantation for the treatment of pancreatic disorders, including diabetes and hyperglycemia.

Expression of the stem cell marker CD133 correlates with the ability of cells to engraft for long periods of time, and these cells have the innate ability to migrate and pool to the site of injury. It has been determined that the cell compositions disclosed herein migrate to the pancreas when injected into STZ-diabetic mice and show normalization of hyperglycemia by a corresponding decrease in blood glucose levels after treatment. For example, six (6) four STZ treated mice were injected with a cell composition comprising (about) 20 x 10 ⁶ cells isolated from murine pancreatic tissue and treated with islet cell medium comprising a polypeptide according to SEQ ID No.1 or 2. The results show that by day 10, all mice had lower blood glucose levels; by day 22 post injection, two of the four animals exhibited fasting blood glucose below 200 mg/dl. Blood glucose levels were reduced in all recipient animals, two of which maintained blood glucose levels near 250mg/dl, with one animal having a level as low as 180mg/dl on day 85 of the experiment. See fig. 5.

Since cells from normal pancreatic tissue exhibit very similar characteristics to cells produced from T1D pancreatic samples, i.e. triple positive for CD133, glucagon and insulin, it is reasonable to expect that a cell composition comprising cells secreting insulin and glucagon produced from a T1D pancreatic tissue sample treated with a islet cell culture medium comprising a polypeptide according to SEQ NO:1 or 2 will provide a measurable decrease in blood glucose levels in vivo. Furthermore, implantation of cell compositions comprising insulin and glucagon secreting cells has been demonstrated to be non-oncogenic, making them a desirable option for treating pancreatic disorders by transplantation (by one of injection, infusion, implantation).

Example 7

Methods for culturing, viability testing and cryopreservation of insulin and glucagon secreting islet cells.

In one embodiment, islet cell culture medium comprises CMRL-1066 (Meditech, #99-663-CV; phenol red free graft medium (CMRL 1066)), supplemented with: 10% heat-inactivated fetal bovine serum (Gibco, # 16140071), 10% human serum (Gemini, # 100512), L-glutamine (2 mM, gibco, # 25030081), ciprofloxacin (2 ml/L, bioworld, # 403100313), amphotericin-B (0.1 mg/L, gibco, # 15290026), penicillin-streptomycin (100,000U/L-100,000. Mu.g/L, gibco, # 10378016) and peptides according to SEQ ID 1 or 2 (3-20. Mu.g/ml, e.g., 3. Mu.g/ml, 5. Mu.g/ml or 10.0. Mu.g/ml). In another embodiment, the cell culture medium used for cryopreservation comprises a cryoprotectant medium (gibco# 12648010). In another embodiment, viability testing is performed using a method comprising using Fluorescein Diacetate (FDA) (SIGMA ALDRICH #F7378) and Propidium Iodide (PI) (SIGMA ALDRICH #P4170).

In one embodiment, human pancreatic tissue is harvested from a human donor suffering from type 1 diabetes or severe pancreatitis via needle biopsy and cultured in vitro in islet cell culture medium comprising CMRL 1066 supplemented with 10% fetal bovine serum, 10% human serum, 2 mmol/L-glutamine, antibiotics, and peptide according to SEQ ID NO 1 or 2 (concentration ranging from 3 μg/mL to 20 μg/mL, e.g. 10.0 μg/mL) on a plate (or bottle) coated with an Adhesion Factor Mixture (AFM) comprising type I collagen and Endothelial Cell Adhesion Factor (ECAF). Different ratios of ECAF and collagen were used, including a 50/50 ratio of collagen to ECAF. A thin layer of AFM (3 to 10 ml) was applied and after 30 minutes of standing the excess AFM was removed; the plates were dried in a fume hood for 45 minutes. The plates were then washed with Phosphate Buffered Saline (PBS) to remove any potential contaminants. Harvested pancreatic tissue was incubated on AFM treated flasks/plates for 10 to 20 days in islet cell culture medium until cells began to migrate and proliferate. When about 70-80% confluence in culture was achieved using standard aseptic techniques in a vertical laminar flow hood, the cultured cells were isolated. The separation technique involves removing the supernatant from the culture plate (preserving the supernatant). The plate was then washed with 2-5ml PBS (preserve wash). The cultured cells were dissociated using about 3-5ml of trypsin (25% solution) by incubating the cells in the presence of trypsin at 37 ℃ for about 3-5 minutes until the cells dissociated. Plates were then washed twice with PBS. The trypsin-treated cells were then centrifuged at 1000rpm for 7 minutes at 4 ℃ along with the stored PBS wash and the collected cell culture supernatant. The resulting supernatant was decanted, the pellet was resuspended in 2ml PBS and centrifuged. The supernatant was then removed, and the pellet was resuspended in islet cell medium comprising SEQ ID 1 or 2 and re-plated at a cell density of about 1000 cells/cm ². Cultured cells not used for re-plating were resuspended in cryopreservation medium at a maximum concentration of 1X 10 ⁶/ml. The cultured cells were centrifuged (1000 rpm for 7 minutes) at 4 ℃. The supernatant was aspirated and replaced with fresh cryopreservation medium, and the cells were transferred to a freezer Mr. Frosy (Thermo-Fisher # 5100-0036) overnight and then transferred and stored in gas phase liquid nitrogen.

The cells were subjected to viability assay prior to cryopreservation. Minimum viability accepted by cryopreservation = 95% (thus 95% of all analyzed cells were stained by FDA (viable fluorescent dye).

In an Eppendorf tube, two aliquots of about 200 cells were transferred in a volume of about 50. Mu.l into 400. Mu.l of a solution containing FDA at a concentration of 0.46. Mu.M and PI at a concentration of 14.34. Mu.M. The cells were centrifuged (1000 rpm for 7 minutes) and approximately 95% of the supernatant was aspirated and the cells were transferred to residual fluid in the microscope slide using a pipette. Cells were analyzed under a fluorescence microscope (Olympus model CKX 3) using a green/red filter set. Living cells stained green (FDA) and dead cells stained red (PI). The percentage of viable cells relative to the total number (expressed as a percentage) was determined independently by two operators.

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 relation to any of the aspects and embodiments described above may be applied interchangeably between different embodiments.

Aspects of the invention

Aspect 1. A composition comprising a population of insulin-and glucagon-secreting cells produced by non-insulin-secreting pancreatic cells harvested from a type 1 diabetic donor pancreas, pancreatitis donor pancreas, or a combination thereof, via needle biopsy.

The composition of aspect 2.1, wherein at least about 50% of the population of insulin and glucagon secreting cells express CD133, glucagon, and insulin.

Aspect 3 the composition of any one of aspects 1-2, wherein about 50% to about 100% of the population of insulin and glucagon secreting cells express CD133, glucagon, and insulin, including all values therebetween, such as, for example, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%; the percentages are specified based on the total number of cell populations in the medium).

The composition of any of aspects 1-3, wherein the population of insulin-and glucagon-secreting cells comprises from about 3 x 10 ⁶ cells to about 25 x 10 ⁶ cells/kg human recipient body weight, including all values therebetween, such as, for example, about 4 x 10 ⁶ cells, about 5 x 10 ⁶ cells, about 6 x 10 ⁶ cells, about 7 x 10 ⁶ cells, about 8 x 10 ⁶ cells, about 9 x 10 ⁶ cells, about 10 x 10 ⁶ cells, about 11 x 10 ⁶ cells, about 12 x 10 ⁶ cells, about 13 x 10 ⁶ cells, about 14 x 10 ⁶ cells, about 15 x 10 ⁶ cells, about 16 x 10 ⁶ cells, about 17 x 10 ⁶ cells, about 18 x 10 ⁶ cells, about 19 x 10 ⁶ cells, about 20 x 10 ⁶ cells, about 21 x 10 ⁶ cells, about 22 x 10 ⁶ cells, about 12 x 10 ⁶ cells, about 13 x 10 ⁶ cells, about 14 x 10 x ⁶ cells, about 19 x 10 ⁶ cells, and about 8 x 24 x ⁶ cells; wherein the mass (in kg) of a typical human subject depends on, for example, age and height, and may range from about 4kg to about 225kg, including all values therebetween, such as about 10kg, about 20kg, about 30kg, about 40kg, about 50kg, about 60kg, about 70kg, about 80kg, about 90kg, about 100kg, about 110kg, about 120kg, about 130kg, about 140kg, about 150kg, about 160kg, about 170kg, about 180kg, about 190kg, about 200kg, about 210kg, and about 220kg.

The composition of any one of aspects 1-4, wherein the non-insulin secreting pancreatic cells harvested via needle biopsy are obtained from a type 1 diabetic donor pancreas.

The composition of any one of aspects 1-5, wherein the non-insulin secreting pancreatic cells harvested via needle biopsy are obtained from a pancreatitis donor pancreas.

Aspect 7 the composition of any one of aspects 1-6, wherein the non-insulin secreting pancreatic cells are autologous, allogeneic or a combination thereof.

Aspect 8. A method of treating a pancreatic disorder comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of aspects 1-7, wherein the pancreatic disorder comprises type 1 diabetes, pancreatitis, or a combination thereof.

The method of aspect 9, aspect 8, wherein the administering comprises delivering to the subject a therapeutically effective amount of the composition of any one of aspects 1-7 via one or more of injection, infusion, omentum or peritoneal bag (pouch), surgical implantation, or via delivery of the composition to a target site of the subject as part of a device.

Aspect 10. A method for preparing a composition comprising a population of insulin-and glucagon-secreting cells, the method comprising: treating a population of non-insulin secreting type 1 diabetic pancreatic cells in vitro with a pancreatic islet cell culture medium comprising a basal medium and an effective amount of a polypeptide comprising 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.

The method of aspect 11, aspect 10, wherein the treating further comprises differentiating the non-insulin secreting type 1 diabetic pancreatic cell population and proliferating the insulin and glucagon secreting cell population.

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

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

The method of aspect 14, aspect 10, wherein the treating further comprises differentiating a non-insulin secreting type 1 diabetic pancreatic cell population and proliferating the insulin and glucagon secreting cell population, wherein the insulin and glucagon secreting cell population comprises from about 3 x 10 ⁶ cells to about 25 x 10 ⁶ cells/kg human recipient body weight.

The method of any one of aspects 10-14, further comprising extracting the non-insulin secreting type 1 diabetic pancreatic cell population from the donor.

The method of any one of aspects 10-14, further comprising extracting the non-insulin secreting type 1 diabetic pancreatic cell population from the donor via needle biopsy.

The method of any one of aspects 17, 10-16, further comprising extracting the non-insulin secreting type 1 diabetic pancreatic cell population from an autologous donor and an allogeneic donor, or a combination thereof.

The method of any of aspects 10-17, wherein the islet cell culture medium comprises the polypeptide in an amount in the range of about 3 μg/mL to about 20 μg/mL, and all values therebetween, such as, for example, about 4 μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8 μg/mL, about 9 μg/mL, about 10 μg/mL, about 11 μg/mL, about 12 μg/mL, about 13 μg/mL, about 14 μg/mL, about 15 μg/mL, about 16 μg/mL, about 17 μg/mL, about 18 μg/mL, and about 19 μg/mL.

The method of any one of aspects 10-19, wherein the treating further comprises differentiating the non-insulin secreting type 1 diabetic glucagon cell population and proliferating the insulin and glucagon secreting cell population to obtain a therapeutic amount of the insulin and glucagon secreting cell population, and the method further comprises administering a therapeutically effective amount of the insulin and glucagon secreting cell population to a subject in need thereof.

Aspect 20. A composition (e.g., any of aspects 1-7) comprising a population of insulin-and glucagon-secreting cells produced by non-insulin-secreting pancreatic cells harvested from a type 1 diabetes donor pancreas via needle biopsy, for use in treating type 1 diabetes, pancreatitis, or a combination thereof.

While the foregoing information has been shown for purposes of illustration and example in a highlighted manner, 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 those skilled in the art that the features described in relation to any one of the aspects and embodiments described above may be applied interchangeably between different embodiments.

The aspects and embodiments described above are examples for illustrating various features of the subject matter claimed herein. All publications and patent applications disclosed herein are indicative of the level of skill of those skilled in the art to which the subject matter of the present disclosure and claims pertains.

Throughout the description and claims of this specification, the words "comprise" and "comprising" and variations thereof mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components or steps. Throughout the specification and claims of this document, the singular includes the plural unless the context requires otherwise. In particular, when indefinite articles are used, the context will be understood to consider a plurality as well as a single unless the context requires otherwise.

Features, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example are to be understood as applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed herein (including any 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 where at least some of such combined 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 any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Specific patent applications incorporated by reference include, for example, U.S. patent application No. 15/811,060 (and disclosed as US2018/0133280 A1) filed on the date of 11/13 in 2017; and international patent application number PCT/US2019/038305 filed on date 20 and 6 in 2019 (and published as WO 2020/005721 A1). To the extent that a term and/or expression incorporated herein conflicts with a term and/or expression disclosed herein, the information disclosed herein controls.

Claims

1. A composition comprising a population of insulin-and glucagon-secreting cells produced by non-insulin-secreting pancreatic cells harvested from a type 1 diabetes donor pancreas, pancreatitis donor pancreas, or a combination thereof, via needle biopsy.

2. The composition of claim 1, wherein at least about 50% of the population of insulin and glucagon secreting cells express 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 express CD133, glucagon, and insulin (provided that all values therebetween are included, such as, for example, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%; provided percentages are based on the total number of cell populations of the medium).

4. The composition of claim 1, wherein the population of insulin and glucagon secreting cells comprises from about 1.2 x 10 ⁶ cells to about 25 x 10 ⁶ cells/kg of human recipient body weight (prescribed: including all values therebetween, such as, 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; prescribed generally the mass (in kg) of human recipients (e.g., children (e.g., 4-60 kg) and adults (60-225 kg)).

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

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

7. The composition of claim 1, wherein the non-insulin secreting pancreatic cells are autologous, allogeneic, 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 said pancreatic disorder comprises type 1 diabetes, pancreatitis, or a combination thereof.

9. The method of claim 8, wherein the administering comprises delivering a therapeutically effective amount of the composition of claim 1 to the subject via one or more of injection, infusion, omentum or peritoneal bag, surgical implantation, or via delivery of the composition to a target site of the subject as part of a device.

10. A method for preparing a composition comprising a population of insulin-and glucagon-secreting cells, the method comprising:

treating a population of non-insulin secreting type 1 diabetic pancreatic cells in vitro with a pancreatic islet cell culture medium comprising 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, a polypeptide comprising 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 of claim 10, wherein the treating further comprises differentiating the non-insulin secreting type 1 diabetic pancreatic cell population and proliferating the insulin and glucagon secreting cell population.

12. The method of claim 10, wherein the treating further comprises differentiating the non-insulin secreting type 1 diabetic glucagon cell population and proliferating the insulin and glucagon secreting cell population, at least about 50% of the insulin and glucagon secreting cell population expressing CD133, glucagon, and insulin.

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

14. The method of claim 10, wherein the treating further comprises differentiating the non-insulin secreting type 1 diabetic pancreatic cell population and proliferating the insulin and glucagon secreting cell population, wherein the insulin and glucagon secreting cell population comprises from about 3 x 10 ⁶ cells to about 25 x 10 ⁶ cells/kg human recipient body weight.

15. The method of claim 10, further comprising extracting the non-insulin secreting type 1 diabetic pancreatic cell population from a donor.

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

17. The method of claim 10, further comprising extracting the non-insulin secreting type 1 diabetic pancreatic cell population from an autologous donor and an allogeneic donor, or a combination thereof.

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

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

20. A composition comprising a population of insulin-and glucagon-secreting cells produced by non-insulin-secreting pancreatic cells harvested from a type 1 diabetic donor pancreas via needle biopsy, for use in treating type 1 diabetes, pancreatitis, or a combination thereof.