The application relates to the field of biotechnology, medicine, and cell culture. It specifically relates to, e.g., methods of producing compositions of and comprising cell clusters (also identified as “Neo-Islets” or “NIs”) that include stem cells and pancreatic islet cells (ICs). It also relates to the utilization of cell clusters comprising stem cells and pancreatic islet cells for treatment of, for example, insulin-dependent diabetes mellitus, noninsulin-dependent diabetes mellitus, or impaired glucose tolerance.
Insulin-producing β-Cells, when isolated from a donor pancreas, generally proliferate very poorly ex vivo, i.e., not sufficiently to generate adequate cell numbers for the treatment of insulin-dependent diabetes mellitus. Current technologies and many preclinical therapies designed to overcome this shortage and provide diabetic patients with a long-lasting, physiologically released insulin replacement therapy (islet and pancreas transplants; precursor cell-derived therapies, etc.) are hampered both by the shortage of donor cells and the need to suppress the patient's immune system, leading to a new set of adverse effects for the patient, such as opportunistic infections and malignancies. The great shortage of suitable pancreas donors combined with the need for repeated islet transplants, requiring up to five donors each, continues to prevent the general availability of these expensive therapies. Micro- and macro-encapsulation systems of insulin-producing cells are tested to facilitate immune isolation and overcome this problem. However, the utilized encapsulation materials represent foreign bodies and can induce a foreign body reaction that will result in the failure of the therapy or require use of anti-rejection drugs if the encapsulation device is open.
Described herein are methods of making cell clusters, the method comprising: expanding pancreatic islet cells; and forming cell clusters comprising: the expanded pancreatic islet cells; and mesenchymal stem cells and/or adipose stem cells.
In embodiments, expansion of the pancreatic islet cells includes at least five population doublings before forming the cell clusters.
In embodiments, the pancreatic islet cells are primary pancreatic islet cells obtained from an adult donor; and wherein the adult donor's islets had a North America Islet Donor Score (NAIDS) of less than 80.
Further described herein are cell clusters produced by the method described herein.
Also described are methods of treating a subject, the methods comprising: providing to the subject the cell clusters described herein. Additionally, described are methods of treating a subject suffering from Type 1 Diabetes Mellitus or Type 2 Diabetes Mellitus, by, e.g., providing to the subject cell clusters as described herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The disclosed methods, cells, and cell clusters overcome the limited ability to generate sufficient therapeutic doses of isolated and cultured pancreatic islet cells from a single pancreas donor and provide them to a subject in need thereof.
As used herein, Islets may comprise any of the cells found in mammalian pancreatic islets, including but not limited to Alpha cells, Beta cells, Delta cells, Gamma cells, and Epsilon cells. In one embodiment Islets comprise at least insulin expressing Beta cells.
As used herein, cell clusters may comprise Bone Marrow-derived Mesenchymal Stem Cells and/or Adipose-derived Stem Cells, and expanded pancreatic islet cells. The expanded pancreatic islet cells may be dedifferentiated pancreatic islet cells and/or redifferentiated pancreatic islet cells. The redifferentiated pancreatic islet cells may comprise any of the cells found in mammalian pancreatic islets, including but not limited to Alpha cells, Beta cells, Delta cells, Gamma cells, and Epsilon cells. Thus, the cell clusters hereof preferably produce, among other things, insulin, glucagon, somatostatin, pancreatic duodenal homeobox-1, insulin transcription factor mafA, nk6 homeobox-1, etc., which helps to better regulate glucose levels and thus explain the surprisingly good results attained herein. In one embodiment, the cell clusters comprise at least insulin-expressing Beta cells. The cell clusters of the present disclosure may comprise, by way of nonlimiting examples, a ratio of dedifferentiated pancreatic islet cells and/or redifferentiated pancreatic islet cells to adipose stem cells and/or mesenchymal stem cells of 1000:1, 100:1, 50:1, 25:1 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:25, 1:50, 1:100, or 1:1000.
Embodiments include cell clusters, generated in vitro, which are the approximate size of pancreatic islets. Such cell clusters may comprise Bone Marrow-derived Mesenchymal Stem Cells (MSCs) and/or Adipose-derived Stem Cells (ASCs); and expanded pancreatic islet cells. The expanded pancreatic islet cells may be dedifferentiated pancreatic islet cells and/or redifferentiated pancreatic islet cells. Culture expansion may dedifferentiate the pancreatic islet cells via Epithelial-Mesenchymal Transition (EMT), and the resulting cells may be aggregated with MSCs and/or ASCs into the cell clusters, which will spontaneously redifferentiate and resume regulated insulin secretion when administered to subjects. Pancreatic islets, like all organs, possess small numbers of MSCs and/or ASCs that intrinsically, as pericytes, exert robust anti-inflammatory, complex immune-protective, pro-angiogenic, survival and tissue repair-supporting actions. Cell clusters containing dedifferentiated cells may be treated to cause redifferentiation, the redifferentiation resulting in cell clusters comprising redifferentiated pancreatic islet cells that express insulin. In vitro creation of cell clusters, composed of culture expanded pancreatic islet cells and much higher numbers of healthy MSCs and/or ASCs than is physiologic, enable these cell clusters, mediated by the pleiotropic actions of MSCs and/or ASCs, to withstand inflammatory, immune and other insults when administered to subjects with impaired glycemic control, such as seen in Type 1 Diabetes Mellitus, Type 2 Diabetes Mellitus, and other types of insulin-dependent diabetes mellitus, or impaired glucose tolerance.
Isolated pancreatic islet cells (primary pancreatic islet cells) may be from any suitable donor (e.g., rodent, canine, human, or other mammal). In embodiments, the donor is an adult donor.
Islets cells may be obtained from demographically diverse pancreas/islet donors or isolated islets that are not suitable for therapeutic use under the current criteria in use by the medical community—referred to herein as “research grade” islet cells. Islet cells from such donors are generally wasted because they are judged unsuited for an islet transplant. However, cells from such donors, when formed into cell clusters as described herein, are suitable for therapeutic use. In short, the methods and cells clusters described herein provide a major expansion of the size of the donor pool from diverse demographic origin (dog and human) and isolated islets from such donors that do not meet the current quality criteria for a successful islet transplant (e.g., lower cell viability).
In aspects, the pancreatic islet cells used here may be classified as “research grade,” i.e., not intended for therapeutic use. In additional embodiments, the pancreatic islet cells may be obtained from a donor having a North America Islet Donor Score (NAIDS) of less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 as defined by Golçbiewska, et al., and Yeh, et al. [4,5]. Specifically included herein are methods for the treatment of subjects with impaired glycemic control, such as Type 1 Diabetes Mellitus, Type 2 Diabetes Mellitus, and other types of insulin-dependent diabetes mellitus, or impaired glucose tolerance by the i.p. administration of the cell clusters described herein where the clusters contain pancreatic islet cells that were expanded from cells classified as research grade or having a NAIDS score as indicated above.
Differentiated pancreatic islet cells express, e.g., insulin, but do not proliferate, or proliferate only minimally in vitro. Isolated pancreatic islet cells may be induced to dedifferentiate in vitro. As used herein, “dedifferentiated” pancreatic islet cells or islet cell nuclei are cells or nuclei that no longer express or produce physiological levels of insulin when challenged with glucose. In certain embodiments, the expression of insulin by dedifferentiated pancreatic islet cells when challenged with glucose may be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to primary isolated pancreatic islet cells. The process of dedifferentiation is also referred to herein as an Epithelial-Mesenchymal transition or an “E to M” transition. Dedifferentiated pancreatic islet cells may proliferate in culture at a rate superior to differentiated pancreatic islet cells. Dedifferentiation of the pancreatic islet cells may immediately reduce or silence insulin expression, insulin synthesis, insulin storage, and/or glucose-induced insulin secretion in these cells.
Dedifferentiated pancreatic islet cells may be allowed to proliferate in vitro to form a large pool of cells that may be co-cultured and/or formed into cell clusters with other cell types.
Proliferation associated dedifferentiation may be achieved by culturing pancreatic islet cells in conditions which are adherent for the pancreatic islet cells. In various embodiments, the pancreatic islet cells may be cultured on a surface that has been coated with or not coated with laminin 511 or laminin 411. Dedifferentiation may optionally be performed in a dedifferentiation medium. Dedifferentiation medium may include a glucagon-like peptide 1 (GLP-1) receptor agonist. In specific embodiments, the GLP-1 receptor agonist may be GLP-1, exenatide, liraglutide, lixisenatide, albiglutide, taspoglutide, and/or Exendin-4. The GLP-1 receptor agonist may be present in the dedifferentiation culture medium at a concentration from 0.1 to 100 nM, from 1 to 50 nM, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, or 30 nM.
In embodiments, the dedifferentiated islets cells may be expanded in culture for at least 1 population doubling prior to inclusion in a cell cluster. Numbers of population doublings that can be undergone include, but are not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, and 50 population doublings before inclusion into a cell cluster.
In particular embodiments, dedifferentiated islets cells may be redifferentiated prior to inclusion in cell clusters described herein.
Culturing of isolated pancreatic islets and/or pancreatic islet cells on laminins (e.g., Laminin 411 and 511), and addition of suitable media, may improve cell adhesion in culture, support cell survival, and moderately boost proliferation. For dedifferentiation, pancreatic islet cells may be plated on a suitable substrate that allows for attachment. In specific embodiments, the substrate may include Laminin 411 and/or Laminin 511. In a more specific embodiment, islet cells may be plated on tissue culture flasks or wells coated with Laminin-411 and/or Laminin-511 and placed in RPMI, DMEM, alpha MEM, CMRL, PIM, or other suitable culture media and supplemented with 10% to 20% fetal bovine serum or other species-specific serum or platelet lysate, and glutamine/penicillin/streptomycin. The culture medium may also be supplemented with at least 10 nM Exendin-4.
Examples of sera in which the cell clusters may be cultured include, but are not limited to, sera available from worldwideweb.sigmaaldrich.com. Specific non-limiting examples include: Fetal Bovine Serum, Bovine Calf Serum, Adult Bovine Serum, Chicken Serum, Goat Serum, Porcine Serum, Rabbit Serum, Sheep Serum, Horse Serum, Canine Serum, Baboon Serum, Coyote Serum, Goose Serum, Mouse Serum, Rat Serum, Rhesus Monkey Serum, Serum Replacement, and Human Serum.
Included are methods of making the cell clusters, the methods comprising: expanding pancreatic islet cells as described herein; and forming cell clusters comprising: the expanded pancreatic islet cells; and mesenchymal stem cells and/or adipose stem cells.
The MSC/ASC component of the cell clusters provides immune isolation, protection, and increased survival of the islet-derived component (the dedifferentiated pancreatic islet cells or redifferentiated pancreatic islet cells), thereby preventing rejection and enhancing engraftment of the cell clusters. Amplification via significantly increased numbers of cells of the potent immune-modulating activities of normal MSCs and/or ASCs in cell clusters provides auto- and allo-immune isolation of pancreatic islet cells, thereby eliminating the need for anti-rejection drugs or encapsulation devices. Consequently, in certain embodiments of treating a subject with the cells described herein, anti-rejection drugs are not administered to the patient. In further embodiments, the cells described herein are not encapsulated and/or associated with an encapsulation device. Moreover, the MSC/ASC component of the cell cluster may induce, via the release of hepatocyte growth and other factors, reversal of the Epithelial to Mesenchymal transition, thus facilitating redifferentiation of dedifferentiated pancreatic islet cells into insulin and other islet hormone producing cells in vivo.
In further embodiments, the cell clusters are administered intra-peritoneally (i.p.). The ability of the mammalian omentum to take up foreign bodies and various cell types facilitates the durable and spontaneous engraftment of the cell clusters, which then deliver insulin to the subject physiologically, i.e., into the portal vein of the liver, additionally optimized by superior peritoneal glucose sensing and oxygen pressures to that in the subcutaneous and portal vein spaces (see, D. R. Burnett, L. M. Huyett, H. C. Zisser, F. J. Doyle, and B. D. Mensh, “Glucose sensing in the peritoneal space offers faster kinetics than sensing in the subcutaneous space,” Diabetes 63:2498-505 (2014), incorporated herein by this reference [6]). The physiological route of insulin delivery might reduce insulin resistance, insulin-enhanced lipogenesis and potentially harmful exposure of peripheral tissues to high concentrations of insulin. For these reasons the omentum is uniquely suited for implantation of the cell clusters, in addition, should the need arise the cell clusters can be removed from the subject via an omentectomy (surgical removal of part or all of the omentum).
In further embodiments, should there be evidence for premature rejection of cell clusters, a short initial course with rapamycin or other suitable anti-rejection agent may administered to the subject to improve cell cluster survival and function. If a recipient of this therapy lacks or has a damaged omentum, an intra portal vein transplant, other location, or a suitable encapsulation device may be utilized.
In each of the above examples of methods, cell clusters may be coated with hydrogel. Such coating may be performed after any step in which a cell cluster is formed or prior to infusing or providing cell clusters to a subject.
In each of the above examples of methods, cell clusters may be contained within an encapsulation device. Such encapsulation may be performed after any step in which a cell cluster is formed or prior to infusing or providing cell clusters to a subject
In various embodiments, the cell clusters may be immune privileged. As used herein, “immune privileged” refers to cell clusters described herein eliciting no or a less robust immune response than cells or cell clusters that are not immune privileged. In various embodiments, the immune response to “immune privileged” cells or cell clusters may be 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or 100% or less than the immune response to non-immune privileged cells or cell clusters.
MSCs and ASCs are undifferentiated, multipotent, adult stem cells, also known as stromal cells that proliferate well, and do not produce insulin. MSCs and ASCs may be from any suitable donor (e.g., rodent, canine, human, or other mammal).
Dedifferentiated pancreatic islet cells proliferate well, but do not, or only minimally express or secrete insulin. In some embodiments, dedifferentiated pancreatic islet cells are allowed to proliferate to generate sufficient numbers for subsequent manipulation. In certain embodiments, once sufficient dedifferentiated pancreatic islet cells have been generated the cells are treated with an islet cell or beta cell-specific redifferentiation medium. Redifferentiation of the pancreatic islet cells restores insulin production, resulting in the re-expression of physiological insulin expression, synthesis, storage, and glucose-sensitive insulin release.
Described is the redifferentiation of dedifferentiated pancreatic islet cells to generate a redifferentiated islet cell. Redifferentiation, as used herein, refers to the treatment of dedifferentiated pancreatic islet cells to generate a redifferentiated islet cell having restored expression of physiological insulin expression, synthesis, storage, and glucose-sensitive insulin release. In certain embodiments, redifferentiation may be a two-step process.
In a first step, a dedifferentiated islet cell may be exposed to a culture medium containing a low level of glucose. The low level of glucose may be selected from 1, 2, 3, 4, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6 mM D-glucose. The medium may contain other components such as Insulin/Transferrin/Selenium (ITS), penicillin/streptomycin (Pen/Strep), fetal bovine serum (FBS), dog serum, or human platelet lysate. The first step may include culturing the dedifferentiated islet cell in the culture medium containing a low level of glucose for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 to 14, 2 to 13, 3 to 12, 4 to 10, or 5 to 9 days.
In a second step, the dedifferentiated islet cell may be exposed to a culture medium containing a high level of glucose. The high level of glucose may be selected from 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 mM D-glucose. The medium may contain other components such as insulin/transferrin/selenium (ITS), penicillin/streptomycin (Pen/Strep), fetal bovine serum (FBS), dog serum, or human platelet lysate, N2 supplement, B27 supplement, nicotinamide, Activin A, Alk-5 inhibitor II, triiodothyronine, and a glucagon-like peptide 1 (GLP-1) receptor agonist. Nicotinamide may be present in the culture medium at a concentration from 0.1 to 100 mM, from 1 to 50 mM, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, or 30 mM. Activin A may be present in the culture medium at a concentration from 0.1 to 100 mM, from 1 to 50 mM, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, or 30 mM. The GLP-1 receptor agonist may be present in the culture medium at a concentration from 0.1 to 100 nM, from 1 to 50 nM, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, or 30 nM. The Alk-5 inhibitor II may be present in the culture medium at a concentration from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, or 30 μM. The triiodothyronine may be present in the culture medium at a concentration from 0.1 to 100 μM. The GLP-1 receptor agonist may be Exendin-4. The second step may include culturing the dedifferentiated islet cell in the culture medium containing a high level of glucose for 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 10 to 28, 11 to 27, 12 to 26, 13 to 25, or 14 to 29 days.
In some embodiments, a method is provided for generating insulin-producing cells through a substantial expansion in the amount of starting material (dedifferentiated pancreatic islet cells) for subsequent culturing with proliferating MSCs or ASCs.
Methods are disclosed for the formation of the cell clusters as described herein. Such cell clusters may be approximately the size of islets found in the pancreas. Cell clusters may be formed, e.g., by any method known in the art. In a non-limiting example, cell clusters are formed by the culturing of cells on hydrophobic, ultra-low adhesion surfaces.
Examples of hydrophobic and/or ultra-low adhesion surfaces include, but are not limited to untreated polystyrene, low attachment hydrogel layers, and uncharged surfaces. Also described are methods of treating a subject in need of insulin and/or suffering from Type 1 (“T1DM”) or Type 2 Diabetes Mellitus (“T2DM”), or suffering from impaired glucose tolerance or Prediabetes Mellitus, using the described cell clusters is disclosed. In some embodiments, cell clusters are administered intraperitoneally (i.p.) and/or to the omentum of the subject. In certain embodiments, cell clusters are administered s.c., or otherwise parenterally to the subject. In certain embodiments, administration of the cell clusters to the subject increases and/or restores insulin production, secretion, and glucose-responsiveness. In certain embodiments, the cell clusters may be coated with hydrogel or other FDA approved material prior to administration to further enhance survival of the cell clusters in vivo, such as gelfoam, or a thrombin clot. In embodiments where the cell clusters contain dedifferentiated pancreatic islet cells, these cells may undergo redifferentiation in the subject after treatment of the subject with the cell clusters.
Methods of treating subjects with cell clusters comprise providing a dose of cell clusters comprising a therapeutically sufficient number of the cell clusters to a subject suffering from T1DM, T2DM, or impaired glucose tolerance to increase and/or restore insulin production, secretion, and glucose-responsiveness. This dose would be understood by those of ordinary skill in the art to vary depending on the route of administration, the weight of the subject, the degree of pathology in the subject to be treated, and the subject's response to therapy. In certain embodiments, subsequent doses of cell clusters could be administered to the subject depending on their initial response to therapy. In embodiments, a therapeutically sufficient number of cell clusters comprises sufficient expanded pancreatic islet cells to increase and/or restore insulin production, secretion, and glucose-responsiveness. In particular embodiments, a therapeutically sufficient number of the cell clusters comprises at least 1.00E+01, 1.00E+02, 1.00E+03, 1.00E+04, 1.00E+05, 1.00E+08, 2.00E+08, 3.00E+08, 4.00E+08, 5.00E+08, 7.00E+08, 8.00E+08, 9.00E+08, 1.00E+09, 2.00E+09, 3.00E+09, 4.00E+09, 5.00E+09, 7.00E+09, 8.00E+09, 9.00E+10, 1.00E+10, 2.00E+10, 3.00E+10, 4.00E+10, 5.00E+10, 7.00E+10, 8.00E+10, 9.00E+10, 1.00E+11, 1.00E+12, 1.00E+13, 1.00E+14, 1.00E+15, 1.00E+16, 1.00E+17, 1.00E+18, 1.00E+19, or 1.00E+20 expanded pancreatic islet cells.
The high efficiency (i.e. the very small loss of viable cells) of the methods described herein also provides a significant increase in the number of doses that can be obtained from a single pancreas over currently conventional treatment. For example, based on the average number of islets that can be obtained from a single human pancreas, expanding the pancreatic islet cells as described herein may provide, from a single donor, sufficient pancreatic islet cells for 10, 25, 50, 75, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 or more doses of cell clusters sufficient to increase and/or restore insulin production, secretion, and glucose-responsiveness. In contrast, current human islet transplants require approximately 3-5 pancreata for a single human dose. Further, repeat doses are often needed to reestablish insulin independence.
“Treating” or “treatment” does not require a complete cure. It means that the symptoms of the underlying disease are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. Insulin requirements may be reduced. End organ damage may be reduced. The need for anti-rejection drugs may be reduced or eliminated. It is understood that reduced, as used in this context, means relative to the state of the disease, including the molecular state of the disease, not just the physiological state of the disease.
The treatment (especially in the early stages) may be aided by the administration of insulin and/or oral hypoglycemic agents (or drugs). Such drugs include the biguanides (e.g., metformin), sulfonylureas (e.g., glimepiride, glyburide, or glipizide), meglitinides (e.g., repaglinide), diphenylalanine derivatives (e.g., nateglinide), thiazolidinediones (e.g., pioglitazone), DPP-4 inhibitors (e.g., sitagliptin, saxagliptin, linagliptin), alpha-glucosidase inhibitors (e.g., acarbose or miglitol), bile acid sequestrants (e.g., colesevelam), etc. Dosages and administration of such drugs, adjuvants and/or intermediate treatment(s) would be readily determined by a person of ordinary skill in the art and dependent on the subject being treated, and need not be repeated here.
Also described are methods of preparation and packaging the cell clusters known in the art to allow for preparation of the cell clusters remotely from the subject to be treated while ensuring survival of the cell clusters before administration, further enhancing survival of the cell clusters in vivo after administration. For instance, various implants are well known to those of ordinary skill in the art. Encapsulation and microencapsulation devices and methods are also well known.
Packaging may be accomplished, for example, by means known in the art, such as packaging fresh or frozen cell clusters into, e.g., syringes, sterile bags, infusion bags, bottles, etc., for delivery to a subject or health care practitioner. Plasmalyte A pH 7.4 maybe extremely useful in packaging the cell clusters.
The use of animal models, including rodent and canine models, is well understood by those of ordinary skill in the art to provide a useful tool in developing treatments for human diabetes [7]. Indeed, as King notes, it is ideal to provide more than one animal model to better represent the diversity of human diabetes, as is disclosed herein. The description provided would enable those of ordinary skill in the art to make and use cell clusters to treat T1DM, T2DM, and impaired glucose tolerance in humans without any undue experimentation.
In some embodiments, the subject may be a mammal, such as, for example, a rodent, dog, cat, horse, or human. In further embodiments, cells in the cell cluster may be allogenic, xenogenic, or a combination of allogenic and xenogenic cells in relation to the subject or other cells in the cell cluster.
As used herein, “comprising,” “including,” “containing,” “characterized by,” and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method steps, but also include the more restrictive terms “consisting of” and “consisting essentially of.”
The following examples are provided for illustration purposes only and are not to be construed as limiting the disclosure to the embodiments specifically disclosed therein.
Since Pancreatic islets, like all tissues, possess small numbers of Mesenchymal Stem Cells, as pericytes, that exert immune-modulating, anti-inflammatory and other protective trophic effects locally, [8-14] we hypothesized and tested whether cell clusters (Neo-Islets (NIs)) comprising endocrine pancreatic islet cells with much higher numbers of MSCs/ASCs could be formed, and whether such cell clusters would provide effective: (i) Auto- and/or allo-immune-Isolation without encapsulation devices, (ii) Survival benefits of allogeneic cell clusters in vivo, thereby reducing or eliminating the need for anti-rejection drugs, (iii) Redifferentiation in vivo of pancreatic islet cells, and thereby (iv) Adequate and physiologic insulin secretion and durable maintenance of euglycemia in rodents with T1DM.
The unique and well documented pleiotropic and largely comparable actions of bone marrow-derived Mesenchymal Stromal Cells (MSCs) or adipose tissue-derived Adipose Stem Cells (ASCs), if combined with equal numbers of pancreatic islet cells in islet-sized cell clusters or “cell clusters” (NI), are harnessed to shield administered β-cells from allo- and auto-immune attacks and inflammatory damage, and to enhance β-cell survival and induce angiogenesis. Physiologically, only about 2% of the total cell numbers in islets are MSCs, located as pericyte-like cells in microvascular niches. Their cytoprotective functions within the islets likely parallel those in the bone marrow and other organs, i.e., vasculo-protection and stabilization, anti-inflammatory, trophic and immune-modulating activities. Such NIs of approximate islet size were generated in vitro from culture expanded, via Epithelial-Mesenchymal Transition (EMT) and associated dedifferentiation, pancreatic islet cells and bone marrow-derived MSCs of C57Bl/6 mice. 5×103 NIs, each composed of ˜500 pancreatic islet cells and ˜500 MSCs, were intraperitoneally (i.p.) administered to spontaneously diabetic, immune-competent NOD mice that develop an auto-immune form of T1DM that largely resembles human T1DM. This allogeneic treatment protocol was chosen as it models the most common clinical situation in recipients of pancreas or islet transplants. By not using anti-rejection drugs or encapsulation devices, we rigorously tested that high numbers of MSCs in NIs do enable pancreatic islet cells to survive, redifferentiate into normally functioning endocrine cells, and thereby durably establish glycemic control in NOD mice with autoimmune T1DM. While NI treated diabetic NOD mice thrived normally, vehicle treated, diabetic NOD mice remained hyperglycemic and began to die. These initial data implied that NIs survive, engraft in the omentum, and redifferentiate into functional endocrine cells in vivo, and that both allo- and auto-immune protection is achieved. Importantly, following i.p. administration the NIs were taken up by the omentum where they engrafted long term and redifferentiated into physiologically insulin- and other islet-hormone-producing cells. NOD mice did not mount a humoral allo-immune response to the MSCs and pancreatic islet cells that are used to form NIs. NI-treated diabetic animals showed a significant increase in regulatory T cell (Treg) numbers in their omenta and spleens compared to animals that were treated with islets. When NIs were injected into nondiabetic animals, they also engrafted and survived in the omentum without causing hypoglycemia, further demonstrating regulated insulin secretion. Insulin secretion from the omentum occurs into the portal system of the liver, as does that from the pancreas, which is physiologic and results in inactivation of ˜50% of the delivered insulin. This limits the post-hepatic exposure of muscle, adipose tissue, the vasculature and other organs to supraphysiological, potentially hypoglycemia-inducing and otherwise harmful insulin levels that are generated when insulin is subcutaneously given. When streptozotocin (STZ) diabetic mice were treated with similarly sized cell clusters composed of either only MSCs or Islet cells, blood glucose levels, compared to NI treated, euglycemic animals, were only minimally lowered compared to vehicle treated controls. This clearly demonstrated that the therapeutic efficacy of NIs depends critically on the collaboration of MCSs and pancreatic islet cells. Finally, when STZ-diabetic NOD/SCID mice were treated i.p. by identical protocol with canine NIs (cNI), euglycemia was readily and durably induced and intraperitoneal Glucose Tolerance Tests (IP GTT) were normalized. Importantly, the insulin that was released during the IP GTT was canine specific, and when cNIs were surgically removed, hyperglycemia redeveloped. Taken together, the present data demonstrate that the complex pleiotropic actions of MSCs or ASCs (M/ASCs), as hypothesized, can be readily harnessed to protect cultured pancreatic islet cells, and when combined with them in NIs and administered i.p., facilitate long term glycemic control in mice with autoimmune T1DM. We conclude, therefore, that these observations have significant translational relevance for the treatment of T1DM.
Reagents: Reagents used and their sources are listed in the following table.
From Rodents: Mice were euthanized with Isoflurane (3-5%) in a sealed chamber, and immediately placed on a surgical board for a sterile midline incision. The pancreas was exposed, the pancreatic duct located. The common bile duct was clamped, and the pancreas was inflated with 5 ml/mouse or 15 ml/rat 1 mg/ml Collagenase P in Dissociation Buffer (Hanks Buffered Saline Solution (HBSS), Ca++, Mg+++25 mM HEPES+NaHCO3) via the common bile duct. The inflated pancreas was removed to a sterile conical tube containing digestion solution (1 mg/ml Collagenase P in Dissociation Buffer.). The tube was placed in a 37° C. shaking water bath (120 rpm) and the contents digested for 15 minutes. The digestion was stopped with an equal volume of cold Dissociation Buffer. The digested tissue was filtered through a 400 μm screen into a fresh tube, and centrifuged at 1200 rpm for 2 minutes at 4° C. with the brake off. The pellet was washed with 20 ml Dissociation Buffer and centrifuged again (1200 rpm for 2 minutes at 4° C. with the brake off). To purify the islets further, the pellet was resuspended in 10 ml Histopaque 1077 solution and overlayed with 10 ml serum free DMEM-F12 to set up a gradient. The gradient was centrifuged at 2000 rpm for 20 minutes at 4° C. with the brake off, and the islets were collected at the interface between the medium and Histopaque into a 50 ml conical tube containing 20 ml Dissociation Buffer. The islets were then centrifuged at 1200 rpm for 2 minutes, washed with 20 ml Dissociation Buffer, spun down again, resuspended in islet culture medium, and placed in a sterile Petri dish. Islets were allowed to recover in a 37° C., 5% CO2 humidified incubator at pH 7.4 overnight.
From Dogs: Fresh pancreata were obtained from euthanized dogs through an NIH sharing agreement and inflated via the common bile duct, using 1 mg/ml Collagenase P solution. Canine islets were isolated from inflated pancreases following modified versions of techniques described by Vrabelova, et al. and Woolcott, et al.[15,16] In brief, the distended dog pancreas was cut in 15 to 20 pieces and placed in a 50 ml tube containing 20 ml of 1 mg/ml Collagenase P solution. The tube was placed into a 37° C. water bath with the shaker set at 120 rpm. Islet content in the solution was monitored by microscopic examination of dithizone stained samples obtained from small samples taken at 5-minute intervals. Digestion was continued until approximately 50% of islets were free of acinar tissue, and stopped with 20 ml of HBSS supplemented with 10 mM HEPES+1% BSA. The tissue was then gently sieved through a 400-μm screen and centrifuged for 10 seconds at 100×g at 4° C. The pellets were washed once and centrifuged for 10 seconds at 200×g (4° C.). Three layer density gradients were created by resuspending the pellets in 10 ml Histopaque-1.119, slowly layering on top 10 ml of Histopaque-1.077 followed by another layer of 10 ml of serum-free medium. The gradient was spun at 750×g for 20 minutes at 4° C. without brake. Islets were collected from the top interface and transferred to a 50 ml tube containing HBSS supplemented with 10 mM HEPES+1% BSA. The purified islet suspensions were washed with serum-free medium and centrifuged for 10 seconds at 200×g (4° C.) twice and passed through a 40-μm cell strainer. Five 50 μl aliquots from each preparation were collected and used to assess the islet yield Finally, hand-picked (to remove acinar cell content) islets were cultured in 20% FBS supplemented RPMI 1640 medium at 37° C., in a 5% CO2 incubator.
From Humans: Human islets were purchased from Prodo Laboratories (Irvine, CA) or obtained from other legitimate sources of human donor tissue.
Rodent Islet Cells: Recovered mouse islets were hand-picked and further purified by capturing the islets in the top of a 40 μm filter strainer. Islets were cultured as follows: pancreatic islet cells were cultured by placing whole islets on Laminin-511 coated wells, and allowing the pancreatic islet cells to outgrow from the islets until 90% confluent in RPMI 1640+20% FBS+GPS, which results in their dedifferentiation via reversible EMT. Culturing in this manner further purifies pancreatic islet cells and removes remaining exocrine cells. Passaging: Mouse pancreatic islet cells were allowed to grow to approximately 90% confluence. They were then trypsinized (1× Trypsin-EDTA for 5-10 minutes), pelleted by centrifugation at 600×g for 5 minutes, washed with DMEMF12+20% FBS+GPS, and seeded into T75 flasks. Passaged pancreatic islet cells were cultured in DMEM-F12+20% FBS+GPS. Culturing in this manner further purifies pancreatic islet cells and removes acinar and ductal cells.
Canine Islet Cells: Initial Culture: Recovered dog islets were handpicked and further purified by capturing the islets in the top of a 40-μm filter strainer. Cells were cultured as whole islets as described above for mice. Passaging: see as above for rodent pancreatic islet cells.
Human Islet Cells: Cells were cultured as whole islets and passaged as described above for rodents.
ASCs (mouse and canine): Under sterile conditions, approximately 3-15 g abdominal fat samples were harvested from euthanized, non-diabetic mice or non-diabetic dogs (NIH tissue sharing agreement) and placed on ice in separate, sterile 50 ml conical tubes containing approximately 30 ml of 1×PBS. The fat samples were minced, placed in tubes of PBS containing 3 mg/ml Collagenase 1, and digested approximately 1 hour in a 37° C. shaking water bath. The tubes were centrifuged (600×g, 10 minutes) to pellet the cellular content. The supernatant was carefully removed, and the pellet washed two times with sterile PBS, and then resuspended in 10 ml DMEM F12+GPS+10% FBS for culture. Cells were cultured in a 37° C. humidified 5% CO2 incubator at pH 7.4. Culture medium was changed twice weekly. When primary cultures reached 70-80% confluence, attached cells were passaged by exposure to 1× trypsin/EDTA for 3-5 minutes, and further passaged or cryopreserved in 10% DMSO.
Non-diabetic Human ASCs were purchased at P1 from Lonza (Walkersville, MD), and cultured as described above.
MSCs (from rodents): Obtained cell suspensions from flushed femurs of euthanized mice were plated in T25 flasks containing DMEM-F12+10% FBS+GPS. Cells were cultured in a 37° C. humidified 5% CO2 incubator. Culture medium was changed twice weekly. When primary cultures reached 70-80% confluence, cells were detached with 1× trypsin/EDTA for 3-5 minutes, and passaged or cryopreserved in 10% DMSO.
Prior to cell cluster formation, cultured MSCs or ASCs are characterized (i) by FACS for their expression of CD44 and CD90, and negative expression of CD45, CD34 and DLA-DR antigens, and (ii) by their abilities to undergo trilineage differentiation (adipogenic, osteogenic, chondrogenic) as previously described.[17] Prior to cell cluster formation, cultured, dedifferentiated canine pancreatic islet cells are examined by (a) FACS and confirmed to be negative for expression of DLA-DR, CD90 and CD133; and (b) rtPCR for residual islet cell gene expression of insulin, glucagon, somatostatin, pancreatic polypeptide, pdx-1, and nkx6.1. Cell viability was assessed using Fluorescein diacetate (FDA) and Propidium Iodide (PI) as follows: 1× staining solution (1 μL of 5 mg/ml FDA and 5 μL of 1 mg/ml PI dissolved in 100 μL PBS) was mixed with cells in 100 μL PBS, incubated at room temperature for seconds and cells were imaged using a fluorescence microscope. Four fields were counted for red, green and total cell numbers.
Canine ASCs were tested at P2 for induction of IDO-1 in response to canine interferon gamma (IFNγ) as follows. Eight 35 mm culture dishes were seeded with 0.5×106 canine-derived ASCs each in DMEM F12+10% canine serum. 10 ng/ml canine INFγ was added to four dishes. After overnight culture in a 37° C. humidified 5% CO2 incubator, cells from all dishes were harvested and assayed for IDO-1 gene expression by rtPCR. Results from IFNγ treated cultures were normalized to those of unexposed cells of the same passage number and expressed as Log 10RQ.
Rationale: (A) To test whether cell clusters comprising (i) dedifferentiated, culture expanded pancreatic islet cells combined with (ii) much higher numbers of MSCs/ASCs than occurs naturally in islets could be formed. (B) To determine whether and to what extent such cell clusters express or can be induced to express islet cell associated genes.
Methods
Outgrowth of pancreatic islet cells: Islet cells were either (1) dissociated with trypsin and cells plated in Laminin-511 and/or Laminin-411 (20 μg/ml) pre-coated Tissue Culture (TC) wells or flasks, or (2) whole islets were plated in Laminin-511 and/or Laminin-411 coated TC wells. See
Cell cluster formation: ASCs (P1 to P4) or MSCs (P1 to P5) and Islet cells (P1 to P2) were co-cultured at a 1:1 ratio in ultra-low attachment surface culture dishes (Corning, Kennebunk, ME) and allowed to form NIs overnight. Control ASC and Islet cell clusters were formed by the same method. Prior to their in vivo administration, samples of NIs were tested by rtPCR for expression of islet and MSC associated genes (see below).
Staining for confocal microscopy: ASCs or MSCs were stained with Cell Tracker Green (green), and passaged pancreatic islet cells were stained with Lipophilic Tracer DiI (red) by following the manufacturers' instructions. Post cell staining, NIs were formed, collected, fixed in 10% formalin, and their nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) prior to confocal microscopy.
Lipophilic Tracer DiR labeling of cell clusters was carried out following the manufacturer's instructions.
Redifferentiation of cell clusters: Re-differentiation of cell clusters was achieved in vitro using commercially available additives, in a two-step process. Step 1: cell clusters of rodent, canine or human origin were cultured for 6-8 days in serum free DMEM containing 5.6 mM D-glucose and supplemented with: (a) 1% BSA fraction V, (b) ITS-G, (c) GPS. Step 2: After 6-8 days, this medium was replaced with Redifferentiation Medium (RDM) and cultured 2 weeks. RDM is DMEM containing 25 mM glucose and supplemented with: (a) N2 supplement A (commercially available), (b) SM-1 supplement (commercially available), (c) 10 mM Nicotinamide (commercially available), (d) 10 nM exendin 4 (commercially available), (e) 2 nM Activin A (commercially available). Redifferentiation tested and confirmed by rtPCR for expression of islet and MSC associated genes as described below.
Cell cluster cellular ratio assessment: For each species (mouse, dog, human), adherent cultures of ASCs and ICs were harvested as described above. ASCs were stained green with cell tracker green in order to be able to distinguish them from ICs. Staining efficiency was assessed by FACS and determined to be >95%. ICs were left unstained. NIs were formed overnight in six-well ultra-low adhesion plates as described above using 0.5×106 ASCs and 0.5×106 ICs per well in 2 ml DMEM/F12+10% FBS. The next day, NIs were collected and dissociated to single cell preparations by 30 minutes incubation with 1 ml Accumax per well. Single cell preparations were then resuspended in 1×PBS+1% BSA and analyzed by FACS (BD FACScan Analyzer, San Jose, California) for percent green (ASC) vs. unstained (IC) cells.
rtPCR: RNA was extracted from 1×106 cell samples using a Qiagen RNeasy Mini Kit, including a DNase digestion step to exclude contaminating DNA, and following the manufacturer's instructions. Reverse transcription was performed using SuperScript II Reverse Transcriptase for 60 minutes at 42° C. Real-time PCR was carried out in duplicate using species-specific TaqMan primers (Applied Biosystems, ABS, Foster City, California) and following the manufacturer's instructions. All reactions were carried out in a total volume of 20 μL with TaqMan Universal Master Mix II with UNG. Reaction conditions were 50° C. for 2 minutes, followed by a 95° C. for 10 minutes start, and 40 cycles of melting at 95° C. for 15 seconds and annealing at 60° C. for 1 minute. All samples were run in duplicate, and the average threshold cycle (Ct) value was used for calculations. The ABS 7500 Real Time PCR System was used to monitor real-time PCR. Relative quantitation (RQ, normalization) of each target gene was calculated with the Ct method using the ABS software provided with the instrument, and by normalization to two internal housekeeping genes, beta actin and beta 2 microglobulin (B2m). RQ was calculated through normalization to external controls as indicated, and by using the software provided with the machine. Results are presented as log 10 (RQ)±log 10 (RQmin and RQmax). Differences greater than log 10 (RQ) 2 or less than log 10 (RQ)-2 were considered significant. Utilized PCR primers are listed in the following table.
Growth and Characterization of ICs and M/ASCs: The NIs' starting materials, i.e., cultured ICs and M/ASCs, were obtained as follows. Freshly isolated islets from non-diabetic mice, dogs and humans were tested for viability, placed in culture, and grown and passaged as described in the above examples. ICs grow out of the islets, proliferate and dedifferentiate as they undergo EMT, a reversible process. Cultured ICs retain residual IC-associated gene expression profiles that decrease with passaging, and exhibit a gene expression pattern distinct from those of M/ASCs (
Cell cluster formation and imaging:
NIs of approximate islet size were prepared by overnight co-culturing of bone marrow-derived MSCs or their adipose-derived analogs ASCs (M/ASCs) with culture expanded murine pancreatic islet cells (ICs) at a 1:1 ratio (found to be optimal) in an ultralow cell adhesion system. An example of this process using mouse cells is shown in
Gene expression profiles and Glucose Stimulated Insulin Secretion of murine, canine and human cell clusters: While these cell clusters do not express significant levels of insulin, as cultured pancreatic islet cells undergo an Epithelial to Mesenchymal transition when cultured, they may be redifferentiated in vitro using the redifferentiation protocol outlined above, such that they re-express islet cell genes. When the two cell types, ICs and M/ASCs, were combined to form NIs as shown in
Conclusion: Taken together, these results indicate (i) that cell clusters of cultured pancreatic islet cells and either MSCs or ASCs can be readily formed in vitro; (ii) that across species (mouse, dog, human), such cell clusters are similar in appearance and gene expression profiles, expressing low levels of islet associated genes; (iii) that across species (mouse, dog, human) such cell clusters are capable of being redifferentiated in vitro to re-express pancreatic endocrine associated genes. Furthermore, these results suggest that these cell clusters may be of therapeutic use in treating insulin dependent and non-insulin dependent diabetic humans or animals.
All studies involving animals were conducted in adherence to the NIH Guide for the Care and Use of Laboratory Animals, and were supervised and approved by an institutional veterinarian and member of the IACUC. Mice and rats were purchased from either Jackson Laboratory (Bar Harbor, ME) or Harlan (Haslett, MI), and were housed at constant temperature and humidity, with a 12:12-hour light-dark cycle in regular, shoebox type caging. Unless otherwise indicated, all mice and rats had unrestricted access to a standard diet and tap water. All mouse experiments were carried out using female C57Bl/6, female NOD or female NOD/SCID mice weighing between 15 and 35 g. All rat experiments were conducted on male Sprague-Dawley rats weighing between 538 and 650 g.
Polydextran Particle Omental Uptake Protocol
Four, 2-year-old Sprague-Dawley rats weighing between 538-650 g were anesthetized and treated i.p. with 5 ml polydextran particles (PDP; sterile sephadex G-25, particle size 87-510 μm) suspended 1:1 in Normal Saline. On Day 7 post administration, animals were sacrificed, and their omenta and other organs were harvested and examined for the presence of PDP.
Diabetes Models
Streptozotocin (STZ): Non-Obese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) and C57Bl/6 mice were made diabetic with 3-5 i.p. doses (1 dose per day) of 50-75 mg/kg b.w. STZ, freshly dissolved in 20 mM citrate buffer, pH 4.5. Mice were considered to be diabetic when their non-fasting blood glucose levels were >300 mg/dL on 3 separate days.
Spontaneous: Female NOD mice develop T1DM spontaneously between 12-20 weeks of age. Mice were considered to be diabetic when their non-fasting blood glucose levels were >300 mg/dL on 3 separate days. Insulin treatment: Where indicated in Results and the figures, insulin was administered to diabetic animals via slow-release, sub-cutaneous insulin pellets (Linbits). Animals were anesthetized with isoflurane, and 1-3 Linbit pellets were inserted just under the skin following the manufacturer's instructions. Tail vein blood glucose concentrations were monitored for several days to ensure animals were neither hyper- nor hypoglycemic.
Blood Glucose Monitoring: In all animal studies, blood glucose concentrations were assessed twice per week via tail vein sampling, and using a OneTouch Ultra 2 glucometer (level of detection, 20-600 mg glucose/dL). Anesthesia: Animals were anesthetized with isoflurane, 1-5%, using an inhalation rodent anesthesia system (Euthanex, Palmer, PA). Rectal temperatures were maintained at 37° C. using a heated surgical waterbed (Euthanex, Palmer, PA).
Treatment of diabetic NOD mice with allogeneic NIs from C57Bl/6 mice: Diabetic NOD mice' blood glucose levels were normalized with Linbits, and NIs, composed of P5 eGFP+ MSCs and P1 pancreatic islet cells (2×105 NIs/kg b.wt. suspended in 0.5 ml serum-free DMEM-F12 medium; N=6) or vehicle (0.5 ml serum-free DMEM-F12 medium; N=6) were sterilely administered i.p., using light isofluorane anesthesia on Day 20 post-Linbit administration. No subsequent exogenous insulin was given in either group. At 10 weeks post NI administration, mice were euthanized, and their omenta, livers, spleens, lungs, kidneys and pancreases were harvested and examined by fluorescence microscopy for the presence of eGFP+ NIs. Sera were also collected to test for an allo-IgG response to the cells that make up the NIs. As a positive control for this test, an additional group of 3 NOD mice was given Linbits and treated i.p. with 2×105 freshly isolated, allogeneic islets/kg b.wt. suspended in 0.5 ml serum-free DMEM-F12. These mice were euthanized 14 days post-islet administration, and their sera harvested and examined as above.
STZ diabetic C57Bl/6 mouse treatment with syngeneic NIs, ASC-clusters or IC-clusters: Four groups of 10-week old, STZ-diabetic, blood glucose controlled (via Linbits) wt C57Bl/6 mice were administered i.p. (i) 0.5 ml vehicle (serum-free DMEM-F12; N=6), or 2×105/kg b.wt. (ii) freshly formed NIs (P5 eGFP+ MSCs and P1 ICs; N=6), (iii) clusters composed of P1 ASCs only (N=5), or (iv) clusters composed of P1 ICs only (N=5). Mice were followed as indicated. Upon euthanization, omenta, pancreata, spleens, livers, lungs and kidneys were harvested and fluoroscopically examined for the presence of eGFP+ NIs. In addition, islet associated gene expression profiles were obtained in all omenta and pancreata.
Treatment of non-diabetic mice with mouse or canine NIs: Mouse NIs: Six groups of 2 to 4, 12-week old C57Bl/6 mice each were administered i.p. either (i) 2×105/kg b.wt. freshly formed syngeneic NIs (P5 MSCs and P1 ICs) suspended in 0.5 ml serum-free DMEM-F12, or (ii) 0.5 ml serum-free DMEM-F12 (vehicle). Mice were followed for up to 12 weeks. Canine NIs: Two groups of 9-week old NOD/SCID mice were treated i.p. with (i) 2×105/kg b.wt. freshly formed cNIs suspended in 0.5 ml DMEM/F12 (N=6) or (ii) 0.5 ml serum-free DMEM-F12 (vehicle; N=3). Mice were followed for 10 weeks.
Results
To test our central hypothesis in a clinically highly informative autoimmune TIDM model, we first examined whether the i.p. administration of in vitro generated allogeneic NIs could reestablish euglycemia in spontaneously diabetic NOD mice as a reflection of (i) their survival, (ii) the redifferentiation of pancreatic islet cells contained in the NIs into functional insulin-producing cells in vivo, and (iii) the MSC-mediated cyto-, allo- and auto-immune protection of the transplanted cell clusters.
Treatment of spontaneously diabetic NOD Mice with allogeneic NIs. Since others found that islet progenitor cells and dedifferentiated islet beta cells can differentiate into functional endocrine cells in vivo, we tested whether allogeneic murine NIs as described herein which were administered i.p. to spontaneously diabetic NOD mice, which develop a T-cell mediated, autoimmune form of T1DM, would reestablish euglycemia. This protocol was chosen because it closely resembles the most common clinical situation in which a patient with T1DM receives an allogeneic pancreas or islet transplant. To facilitate both in vivo tracking and post-mortem localization, administered NIs were dually labeled with DiR and composed of P5 MSCs derived from C57Bl/6 mice transgenic for the eGFP gene, constitutively expressed in all tissues, and P1 ICs from wild type C57Bl/6 mice (see
These mice were then divided into two groups and treated i.p. either with 2×105/kg b.wt. NIs from allogeneic C57Bl/6 mice (N=6) or with vehicle (N=6;
Together, these data show that (i) the NIs engraft and survive, (ii) the ICs within the NIs redifferentiate in vivo, providing the mouse with a new, endogenous source of insulin, and (iii) the MSCs contained in the NIs effectively provide cyto-protection and allo- and auto-immune-isolation of the insulin producing cells in NOD mice, and apparently establishing glycemic control in in this clinically highly relevant T1DM model.
Collaboration of Islet Cells and M/ASCs within NIs is Essential to Establishing Normoglycemia in Diabetic Animals. To further explore the collaboration between ICs and M/ASCs in NIs, two experiments were conducted and are summarized in
In vivo Redifferentiation. Data from the NOD mouse experiment (
Rationale: In Example 5, it was shown that freshly formed cell clusters of ASCs and dedifferentiated pancreatic islet cells express low levels of islet associated genes as well as ASC/MSC associated genes. It was also observed that the endocrine derived component of such cell clusters have the capacity to redifferentiate in vitro to re-express higher levels of islet associated genes. Others have shown that endocrine precursor cells can redifferentiate in vivo to produce insulin. We therefore tested (i) whether cell clusters comprising canine ASCs and dedifferentiated pancreatic islet cells can dose-dependently reverse hyperglycemia and affect animal survival, and (ii) whether removal of cell clusters would result in the return of hyperglycemia, confirming that cell clusters are exclusively responsible for the obtained treatment of T1DM.
Methods
cell clusters: cell clusters were formed from canine ASCs (passage 2) and canine cultured pancreatic islet cells (passage 1).
Diabetes Model: Non-obese diabetic/Severe Combined Immunodeficiency (NOD/SCID) mice were made diabetic with 5 i.p. doses of 50 mg/kg body weight Streptozotocin (STZ) in citrate buffer. Once blood glucose levels were >300 mg/dL on 3 separate days, they were given, on Day 0, one slow-release insulin pellet s.c. each (Linbit, Linshin, Canada) in order to control blood glucose levels and thereby avoid glucotoxic cell damage. These pellets expire by approximately 36 days (see
Intraperitoneal Glucose Tolerance Tests (GTT): At 55 days post treatment, 3 vehicle-treated and 5 canine cell cluster-treated mice were fasted 5 hours, whereupon baseline blood glucose levels were assessed using a OneTouch Ultra 2 Glucometer (Johnson and Johnson, New Brunswick, NJ; level of detection limit of 20 to 600 mg glucose/dL) Animals were then anesthetized, and 2 g glucose/kg bw (dissolved in serum free medium and filter sterilized) were administered via i.p. injection under isoflurane anesthesia. Blood glucose levels were assessed at 30 minutes, 60 minutes and 120 minutes post glucose administration.
Treatment Protocols
NOD allogeneic treatment: Once female NOD mice were confirmed to be hyperglycemic (non-fasting blood glucose >300 mg/dL on 3 separate days), they were treated s.c. with Linbit pellets. Once animals' blood glucose levels were normalized they were anesthetized (isoflurane), and cell clusters, composed of P5 gfp+ MSCs and P1 pancreatic islet cells (2×105 cell clusters/kg bw suspended in 0.5 ml serum-free DMEM-F12 medium; n=6) or vehicle (0.5 ml serum-free DMEM-F12 medium; n=3), were administered i.p. No subsequent exogenous insulin was given to animals in either group. Blood glucose levels and body weights were assessed twice per week, and mice were followed for 10 weeks. At 10 weeks post cell cluster administration, animals were sacrificed, and their sera, omenta, livers, spleens, lungs and kidneys and pancreases were harvested, and examined for the presence of cell clusters and insulin. None were found anywhere but the omenta.
C57Bl/6 syngeneic treatment of STZ diabetic animals: STZ-diabetic, blood glucose controlled (via Linbits) C57Bl/6 mice were anesthetized and administered i.p. either (a) 2×105 freshly formed gfp+ mouse cell clusters (P5 gfp+ MSCs and P1 pancreatic islet cells) stained with DiR and suspended in 0.5 ml serum free DMEM-F12 (vehicle; n=3), or (b) 2×105 freshly formed gfp+ mouse cell clusters stained with DiR and embedded in Gelfoam (n=3). Control group animals were anesthetized and treated i.p. with 0.5 ml vehicle (n=3). Blood glucose levels and weights were assessed at baseline and then twice per week in all animals for 18 weeks. Once per week, the animals were examined under isofluorane anesthesia using a Licor, Pearl Impulse imager to track the cell clusters. Upon sacrifice, omentum, pancreas, spleen, liver, lungs and kidneys were harvested and examined for the presence of cell clusters. None were found anywhere but the omenta.
Treatment of non-diabetic mice: Mouse cell cluster administration: Six groups of 2 to 4 non-diabetic, 12 week old female C57Bl/6 mice (average weight of 21.9 g) each were anesthetized and administered i.p. either (a) 2×105 freshly formed mouse cell clusters (P5 gfp+ MSCs and P1 pancreatic islet cells) suspended in 0.5 ml serum-free DMEM-F12 (5 groups sacrificed at different time points for tracking purposes), or (b) 0.5 ml serum free DMEM-F12 (vehicle; 1 group). Blood glucose levels and weights were assessed at baseline and then twice per week for up to 12 weeks. Canine cell cluster administration: Two groups of non-diabetic, 9 week old, female NOD/SCID mice weighing 19.7 to 24.8 g were anesthetized administered i.p. either (a) 2×105 freshly formed, DiR stained canine cell clusters suspended in 0.5 ml DMEM/F12 (N=6) or (b) 0.5 ml serum free DMEM-F12 (vehicle; n=3). Blood glucose levels and weights were assessed at baseline and then twice per week for 10 weeks. Once per week, the animals were examined under isofluorane anesthesia using a Li-Cor, Pearl Impulse imager to track the cell clusters. Upon sacrifice, omentum, pancreas, spleen, liver, lungs and kidneys were harvested and examined for the presence of cell clusters. None were found anywhere but the omenta.
NOD/SCID recent onset diabetes, xenogeneic treatment: Groups of female, 20 week old, STZ-diabetic NOD/SCID mice weighing 17-29 g (n=5 per group) whose blood glucose levels were controlled with Linbit pellets were anesthetized and treated i.p. with (a) 2×105 or (b) 8×104 freshly formed, unredifferentiated canine cell clusters/kg bw embedded in Gelfoam, or (c) vehicle (DMEM/F12). Cell clusters were composed of P1 Islet cells and P2 canine ASCs. Blood glucose levels and body weights were assessed twice per week, and mice were followed for 13 weeks. IP GTTs were performed in the high dose group at 55 days post treatment, and cell clusters were surgically removed from the high dose group of mice in week 10.
NOD/SCID remote onset diabetes, xenogeneic treatment: 11 week old female NOD/SCID mice weighing 18.4 to 22.8 g were made diabetic with three i.p. doses of 75 mg/kg body weight STZ in citrate buffer. The diabetic state was confirmed by blood glucose levels of >300 mg/dL on 3 separate days. Once the animals were confirmed to be diabetic, their blood glucose levels were controlled for approximately 3 months with insulin therapy using s.c. linbit pellets. To confirm that all animals were still diabetic prior to cell cluster or vehicle administration, Linbits were allowed to expire, and mice to re-develop hyperglycemia. Mice were again treated with Linbits (Day 0) to control blood glucose levels and prevent glucotoxic cell cluster damage. Anesthetized mice were then treated i.p. with either (i) 2×105 cell clusters/kg b.w. embedded in gelfoam or (ii) vehicle (0.5 ml DMEM/F12). Cell clusters were composed of P1 Islet cells and P2 canine ASCs. Blood glucose levels and body weights were assessed twice per week, and mice were followed for 11 weeks. IP GTTs: At 55 days post treatment, 3 vehicle-treated and 5 cell cluster-treated mice were fasted 5 hours, whereupon baseline blood glucose levels were assessed. Animals were then anesthetized, and 2 g glucose/kg bw (dissolved in 0.5 ml serum free medium and filter sterilized) were administered via i.p. injection under anesthesia. Tail vein blood glucose levels were assessed at 30 minutes, 60 minutes and 120 minutes post glucose administration.
ELISA for Canine Insulin: Sera from vehicle and cell cluster-treated mice that had been collected during the glucose tolerance tests were examined by ELISA for the presence of canine specific insulin that does not cross react with mouse insulin (Mercodia, Uppsala, Sweden), following the manufacturer's instructions. Sera from a dog, as well as from a C56Bl/6 mouse were also analyzed as positive and negative controls, respectively, for cross-reactivity.
Antibody Response Test: Aliquots of ˜5×104 cells (MSCs, ASCs or pancreatic islet cells that were used to create the cell clusters that were administered) were each incubated with ˜500 μl of serum obtained from cell cluster or canine ASC-treated NOD mice >14 days post cell cluster or ASC administration. The cells were incubated with the sera for 30 minutes at room temperature. After 30 minutes cells were centrifuged at 600×g for 5 minutes, resuspended in FACS buffer and incubated with cy3-conjugated goat-anti-mouse (dilution) IgG antibody. The cells were incubated an additional 20 minutes in the dark at room temperature. One ml 1×PBS+1% BSA was then added, the cells vortexed, centrifuged, resuspended in 400 μl fixation buffer (1% Formaldehyde), and analyzed by FACS (BD FACScan Analyzer, San Jose, CA). A shift of >7% of the cells was considered a positive response, indicating that the serum contained antibodies to the tested cells. Embedding cell clusters in Gelfoam: Individual doses of cell clusters were collected in a 15 ml Falcon tube and centrifuge at 200×g for 2 minutes. The supernatants were discarded, and the pellets resuspended in 0.2 ml serum-free DMEM-F12 each. The cell cluster suspensions were then loaded into 0.5×0.5×0.5 cm blocks of sterile Gelfoam, which were incubated in a 37° C. incubator for 3 hours prior to i.p. administration to mice. Cell cluster embedded in Gelfoam were surgically transplanted under sterile conditions and under anesthesia onto the peritoneal fat-pads and omenta of recipient mice. The abdominal incision was closed with two layer sutures.
In vivo Imaging: In vivo imaging of DiR stained cell clusters was performed in anesthetized mice using the Li-Cor, Pearl Impulse imager.
Results
A dose of 2×105 cell clusters/kg bw administered i.p. 1 month post STZ achieves and maintains euglycemia and promotes animal survival: Three groups of five NOD/SCID mice each were treated i.p. approximately one month after establishment of STZ-induced T1DM diabetes with (a) 200,000 or (b) 80,000 freshly formed, unredifferentiated canine derived cell clusters/kg body weight, suspended in 0.5 ml serum free medium (DMEM-F12), or (c) vehicle (0.5 ml serum free medium). Linbits were given once, ˜1 month after STZ administration (Day 0 in
Intraperitoneal glucose tolerance tests (IP GTTs) were normal in 2×105 cell clusters/kg bw-treated animals, and a rise in blood glucose was accompanied by release of canine insulin: IP GTTs (2 g glucose/kg bw) were performed at 54 days post canine cell cluster treatment (66 days post Linbit therapy) on NOD/SCID mice that had been treated with either the 2×105 canine cell clusters/kg body weight dose or vehicle as described in the Methods. As seen in
Sera from vehicle and cell cluster-treated mice that had been collected during the glucose tolerance tests were examined by ELISA for the presence of canine specific insulin that does not cross react with mouse insulin as described in Methods. As can be seen in
Retrieval of canine cell clusters reestablishes hyperglycemia: On Day 76, the cell clusters were removed from the 2×105 cell clusters/kg bw treatment group. As
Conclusion: The results presented in Example 6 demonstrate that freshly formed canine cell clusters administered i.p. to recent onset diabetic animals redifferentiate in vivo to provide adequate and physiologic insulin secretion and durable, but reversible, maintenance of euglycemia in rodents with T1DM. In addition the ability to remove the clusters via removal of the omentum is a safety feature of this technology when clinically warranted.
Rationale: (A) It is well known that the Omentum accumulates cells and foreign bodies of various sizes. Thus we hypothesized and tested whether the cell clusters when delivered i.p. would be taken up by and engraft in the Omentum. Such a location offers two advantages: (i) As is the case for the pancreas, blood from the Omentum drains directly into the liver via the portal system. Thus insulin and other islet hormones made by the cell clusters would be delivered in physiological fashion. (ii) The Omentum can be removed without significant ill effects, should it be desired for safety or other reasons that the cell clusters be removed. (B) As MSCs and ASCs express potent angiogenic and survival factors, we also examined whether the stem cell component of the engrafted cell clusters enhanced the development of a blood supply for the cell clusters.
Methods
Mouse cell clusters were generated from co-culture in low adherence vessels of P2 Islet cells derived from wild-type C57Bl/6 mice and P5 MSCs derived from C57Bl/6 mice transgenic for the GFP+ gene to facilitate tracking of the cell clusters in vivo. As indicated below, in one group of experiments, after formation, the cell clusters were stained with the Infrared light-excitable carbocyanine probe DiR (Molecular Probes, Eugene, OR) to allow for tracking in vivo.
Dog cell clusters were formed from co-culture in low adherence vessels of P2 dog pancreatic islet cells and P4 dog ASCs that had been stained with DiR to allow for tracking in live animals.
Diabetes model and allogeneic treatment: Female Non-Obese Diabetic (NOD) mice spontaneously develop T1DM at approximately 12-20 weeks of age. Once female NOD mice were confirmed to be hyperglycemic (blood glucose >300 mg/dL on three separate days), they were treated s.c. with Linbit pellets. Once animals' blood glucose levels were normalized, cell clusters (2×105 cell cluster/kg bw suspended in 0.5 ml serum free DMEM-F12 medium) or vehicle (0.5 ml serum free DMEM-F12 medium) were administered i.p. to groups of five animals each. No subsequent exogenous insulin was given to animals in either group. Blood glucose levels and body weights were assessed twice per week, and mice were followed for 10 weeks. At 10 weeks post cell cluster administration, animals were sacrificed, and their sera, omenta, livers, spleens, kidneys and pancreases harvested.
Canine cell cluster administration: DiR labeled dog cell clusters were administered i.p. to 6 NOD/SCID mice, and the mice were examined weekly for 10 weeks under isoflurane anesthesia using a Li-Cor Pearl Impulse™ imager to track the cell clusters.
Syngeneic cell cluster administration: Two syngeneic administration experiments were performed, one in non-diabetic animals, and another in diabetic animals.
Immunohistochemistry: Omenta and other organs were harvested, fixed and embedded as previously described.[18] Omental sections were deparaffinized and stained by immunohistochemistry for DNA with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR) and insulin protein using a guinea-pig anti-insulin antibody (Dako, Carpinteria, CA), and a cy3-conjugated anti-guinea pig antibody (Jackson ImmunoResearch, West Grove, PA) following the manufacturers' instructions.
Results
Cell clusters spontaneously engraft in the murine Omentum and produce Insulin. We hypothesized that injected NIs would home to, attach to, and engraft in the mice' well-vascularized omenta, which would offer the advantage of physiologic insulin secretion into the portal system of the liver. Indeed, as shown in
To further assess the intraperitoneal engraftment pattern and function of DiRlabeled, eGFP+ NIs as detected in
Conclusion: Taken together, the foregoing results demonstrate that across species: (i) cell clusters that are administered i.p. engraft in the omentum where they remain long term, redifferentiate, secrete insulin in physiologic fashion and are not rejected. (ii) The angiogenic properties of the stem cell component of the cell cluster helps vascularize the cell clusters, providing them with needed oxygen, nutrition, and optimized delivery of insulin from the cell clusters into the portal vein of the liver.
Rationale: We showed in Example 5 that the cell clusters are effective in treating recent onset T1DM. We tested here whether cell clusters were also effective in treating remote onset T1DM.
Methods
Cell clusters: cell clusters were formed from canine ASCs (passage 2) and canine cultured pancreatic islet cells (passage 1).
Diabetes Model: Non-obese diabetic/Severe Combined Immunodeficiency (NOD/SCID) mice were made diabetic with 3 i.p. doses of 75 mg/kg body weight Streptozotocin (STZ) in citrate buffer. The diabetic state was confirmed by blood glucose levels of >300 mg/dL on 3 separate days. Once the animals were confirmed to be diabetic, their blood glucose levels were controlled for approximately 3 months with insulin therapy using s.c. linbit pellets. To confirm that all animals were still diabetic prior to the cell cluster or vehicle administration, Linbits were allowed to expire, and all mice re-developed hyperglycemia. Mice were again treated with Linbits (Day 0 on
IP GTTs and Insulin ELISAs—were carried out as described in Example 5, and results were combined with those of animals in Example 6 (recent onset) and presented in
Results
Two groups of 5 diabetic NOD/SCID mice each were treated i.p. at 3 months after STZ-induced T1DM with (a) 200,000 freshly formed canine derived cell clusters/kg body weight suspend in 0.5 ml serum free medium (DMEM-F12) or (b) vehicle. An overview of the experimental design is given in
Conclusion: The above data demonstrate that, as is the case with recent onset diabetes, cell clusters are also effective in establishing euglycemia in remote onset diabetes.
Rationale: We showed in Examples 5 and 7 that canine cell clusters can reverse STZ induced diabetes in NOD/SCID mice. While the NOD/SCID data presented above indicate that cell clusters generated from canine derived cells are capable of safely and effectively undergoing redifferentiation in vivo to produce insulin and secrete it in physiologic fashion long-term, the NOD/SCID model does not address the issues of protection of the transplanted cells from diabetogenic autoimmune and allo-immune attacks. ASCs and MSCs exhibit powerful immune modulating properties.[19] We hypothesized the stem cell component of the cell clusters would provide local immune isolation, and tested whether the cell clusters could restore euglycemia when administered allogenically to spontaneously diabetic NOD mice.
Methods
Mouse cell clusters were generated from co-culture in low adherence vessels of P2 Islet cells derived from wild-type C57Bl/6 mice and P5 MSCs derived from C57Bl/6 mice transgenic for the GFP+ gene.
Spontaneous diabetes model and allogeneic treatment: Once female NOD mice were confirmed to be hyperglycemic (blood glucose >300 mg/dL on 3 separate days), they were treated s.c. with Linbit pellets. Once animals' blood glucose levels were normalized, cell clusters (2×105 cell cluster/kg bw suspended in 0.5 ml serum free DMEM-F12 medium) or vehicle (0.5 ml serum free DMEM-F12 medium) were administered i.p. to groups of 5 animals each. No subsequent exogenous insulin was given to animals in either group. Blood glucose levels and body weights were assessed twice per week, and mice were followed long term.
Results
A dose of 200,000 allogeneic cell clusters/kg bw administered i.p. achieves and maintains euglycemia in spontaneously diabetic NOD mice. Blood glucose levels of vehicle and cell cluster-treated NOD mice are shown in
Conclusion: These data demonstrate that, like the canine cell clusters, mouse cell clusters (i) redifferentiate in vivo to provide adequate insulin secretion to reestablish and maintain euglycemia, and importantly (ii) that they afford immune isolation against both allo- and auto-immune attacks without encapsulation, as hypothesized.
Rationale: From the previous examples, it is apparent that the cell clusters engraft in the omentum where they redifferentiate to produce and secrete insulin. However, if insulin secretion were to be constitutive and non-physiologic, this could potentially lead to episodes of hypoglycemia. We tested, therefore, whether administration of cell clusters to non-diabetic animals would result in hypoglycemia.
Methods
Mouse cell clusters were generated from co-culture in low adherence vessels of P2 Islet cells derived from wild-type C57Bl/6 mice and P5 MSCs derived from C57Bl/6 mice transgenic for GFP+ gene.
Dog cell clusters were formed from co-culture in low adherence vessels of P2 dog pancreatic islet cells and P4 dog ASCs.
Mouse cell cluster administration: Six groups of 2 to 4 non-diabetic C57Bl/6 mice each were administered i.p. either (a) 2×105 freshly formed mouse scell clusters suspended in 0.5 ml serum free DMEM-F12 (5 groups), or (b) 0.5 ml serum free DMEM-F12 (vehicle; 1 group). Blood glucose levels (OneTouch Ultra 2 glucometer) and weights were assessed at baseline and then twice per week for up to 12 weeks.
Canine cell clusters: Two groups of NOD/SCID mice were administered i.p. either (a) 2×105 freshly formed dog cell clusters (N=6) or (b) 0.5 ml serum free DMEM-F12 (vehicle; N=3). Blood glucose levels (OneTouch Ultra 2 glucometer) and weights were assessed at baseline and then twice per week for 10 weeks.
Results
Cell clusters do not cause hypoglycemia in non-diabetic mice. As shown in Example 8 and
Conclusion: These data demonstrate that engrafted cell clusters formed from either mouse or canine cells release insulin physiologically and not constitutively.
Rationale: The preceding examples indicate the cell clusters described herein may be used allogeneically to reestablish normoglycemia in diabetic animals without rejection. The following study was undertaken to further test whether animals treated allogeneically with cell clusters produce antibodies to either of the cell types that make up the cell clusters.
Methods
Mouse cell clusters were generated from co-culture in low adherence vessels of P2 Islet cells derived from wild-type C57Bl/6 mice and P5 MSCs derived from C57Bl/6 mice.
Antibody Response Test: Test sera were incubated with either: (a) 1×105 gfp+C57Bl/6 MSCs, or (b) 1×105 cultured C57Bl/6 pancreatic islet cells for 30 minutes. Positive control sera were incubated with 1×105 canine ASCs. After incubation with serum, the cells were centrifuged, resuspended in FACS buffer and incubated with Phycoerythrin (PE) labeled anti-mouse IgG antibody (Pharmingen, San Diego, CA). The cells were incubated an additional 20 minutes in the dark at room temperature. One ml 1×PBS (Roche, Indianapolis, IN)+1% BSA (Sigma, St. Louis, MO) was then added. The cells were vortexed, then centrifuged, resuspended in fixation buffer (1% Formaldehyde), and analyzed by FACS (BD FACScan Analyzer, San Jose, CA; 10,000 cells counted).
Results
Sera were obtained from:
Mouse MSCs from cell clusters and mouse pancreatic islet cells from the cell clusters were incubated with the collected sera, and then with Phycoerythrin (PE) labeled anti-mouse IgG antibody. The serum-exposed cells were then analyzed by FACS as described above in Methods to determine whether any IgG antibodies to administered MSCs or pancreatic islet cells were present in the sera of treated mice.
As xenogeneic administration of ASCs is known to elicit an immune response, canine ASCs that had been exposed to sera from NOD mice 14 days post canine ASC administration were incubated with PE labeled anti-mouse IgG antibody, analyzed by FACS, and used as positive controls.
If cell cluster-treated mice had developed an allo-immune response to the MSCs or the pancreatic islet cells in the cell clusters, then the PE-labeled α-mouse IgG antibody would bind to the serum exposed cells, and the cells would appear shifted (PE positive) on FACS analysis. A shift of >7% of the cells (% of PE positive cells) on FACS was considered a positive allo-antibody response.
As shown in
NOD mice do not mount an allo-immune IgG Response to the MSCs and Islet Cells of NIs. To examine whether pancreatic islet cells and MSCs contained in the NIs are protected from a humoral immune attack, we assessed whether sera from normoglycemic, NI-treated NOD mice contained IgG antibodies directed against either the MSCs or cultured ICs that were used to generate the administered NIs. Sera from NI-treated, normoglycemic NOD mice contained neither IgG antibodies directed at MSCs nor at cultured ICs, while the i.p. administration of identical numbers of allogeneic (C57Bl/6), freshly isolated islets used as a positive control, elicited a robust antibody response (
Inhibition of Autoimmune Response. Critical to effectively treating autoimmune T1DM with insulin producing cells is the autoimmune isolation of those cells, and the results presented in
Conclusion: The above data indicate that administration of cell clusters does not elicit an antibody response to either cell type that composes the cell cluster, further supporting the hypothesis that the cell clusters provide immune isolation and eliminate the need for anti-rejection drugs and encapsulation devices.
Our extensive in vitro and in vivo data to date and presented above demonstrate that the treatment of experimental T1DM in mice with syngeneic and allogeneic cell clusters, and cell clusters from multiple species are able to effectively re-establish euglycemia, i.e., treat T1DM, and this during long-term follow-up. No Adverse Events, such as oncogenic transformation or ectopic maldifferentiation of cell clusters were observed. This novel therapy can be used as treatment of insulin-dependent diabetes both in companion animals (dogs, cats) and humans with type 1 diabetes mellitus.
Cell clusters containing human cells are generated as described in the above examples using ASC and/or MSCs from human subjects identified as healthy and not suffering from insulin-dependent Diabetes Mellitus and pancreatic islet cells from an allogeneic source.
The purpose of this study was to determine whether human cell derived cell clusters (hNIs) could reduce or eliminate the need for insulin in diabetic NOD/SCID mice as we previously found for dog cell derived cell clusters (cNIs).[3] Specifically, we set out (a) to determine whether passaged human islet cells (hICs) are characteristically comparable to canine islet cells (cICs), both in gene expression and response to cytokines, etc.; and (b) to determine if human hNIs can durably reduce or eliminate the need for insulin as cNIs and mNIs have been shown to do.[3]
Research grade human islets from 8 non-diabetic human donors (see Table A for demographics and Islet Viability) were purchased in lots of ˜5,000 Islet Equivalents from Prodo Labs (Aliso Viejo, CA). Islet cells derived from this inhomogeneous group of islet donors were expanded by culturing whole islets in tissue culture flasks, using RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA)+10% human Platelet Lysate (hPL; Cell Therapy and Regenerative Medicine, University of Utah, Salt Lake City)+1× L-Glutamine-Penicillin-Streptomycin solution (GPS; Sigma G1146) until ˜90% confluent. For passaging, cells were released with 2× Trypsin (Sigma, St. Louis, MO), pelleted by centrifugation at 600×g for 5 min., washed with DMEM 5 mM glucose (Gibco)+10% hPL+GPS (complete medium), and reseeded at a density of 2×10e5 cells into T75 flasks in complete medium. hICs were characterized by rtPCR for expression of IC specific genes. hIC and cIC doubling times and population doublings (PDLs) were calculated by standard methods.
MSC culture: Human, bone marrow derived MSCs were purchased pre-characterized (tri-lineage differentiation, HLA antigens and surface CD markers) from Lonza (Walkersville, MD) and cultured in complete medium as previously described [2,20,21], and used at Passage 3 for formation of NIs.
Dog islets and cell lines: Utilized dog islets and Adipose Stem Cells (ASCs) from inguinal fat were identical to those used in our ongoing pilot study (INAD 012-776) [3] and all dog cell lines were cultured as previously described [3]. All dogs were non-diabetic mongrels, but some had pacemaker-induced congestive heart failure (see Table B for details). Both islets and adipose tissue were obtained through an NIH Organ Sharing Agreement at the University of Utah.
Islet and cell viability were assessed using Fluorescein diacetate (FDA, Sigma F7378) and Propidium Iodide (PI, Life Technologies P3566) staining, following instructions of the respective manufacturers. Islet viability in percent was quantified in 10 different, homogeneously distributed fields of ˜400 human and canine islets. This method does not detect potential apoptotic cell loss.
Human MSCs and canine ASCs and respective ICs were co-cultured in complete medium at a 1:1 ratio in ultra-low adhesion surface culture dishes (Corning, Kennebunk, ME), resulting in highly efficient cell cluster (hNI) and (cNI) formation overnight, as previously reported [2,3].
Population Doubling Times: We previously observed that PDLs of cultured mouse and dog islet cells between different donors vary significantly. However, this inter-donor variability did not affect subsequent function of either mouse or dog cell clusters in vivo. In the present study, doubling times of hICs and cICSs over 4-12 population doublings (PDLs) are shown in
GSIS and Gene Expression Profiles of Insulin and other Islet-specific Hormones as a Function of Population Doublings (PDLs): For the ongoing cell cluster treatment of diabetic dogs, insulin and other islet hormone gene expression levels are assessed in cultured cICs prior to cell cluster formation, and insulin gene expression is used as a measure of potency and serves as release criterion for cICs and canine cell cluster. In order to determine whether hICs (n=6) and cICs (n=6) that are being culture expanded per identical protocol are functionally comparable, we systematically assessed in the cell lines from both species the gene expression levels of insulin (INS), glucagon (GCG), somatostatin (SST), and pancreatic polypeptide (PPY) by rtPCR (see Table C for rtPCR primers), and plotted these as a function of PDLs (see Table D for details). As shown in
We previously reported that culture expanded mouse and dog ICs and cell clusters secrete insulin in response to glucose stimulation, albeit at significantly reduced levels compared to freshly isolated, whole islets [2,3]. Furthermore, dog cell clusters implanted into streptozotocin-diabetic NOD-SCID mice durably induced euglycemia. When retrieved 9 weeks later, these canine cell clusters secreted 15-fold higher concentrations of canine insulin in response to glucose stimulation than did freshly formed dog cell clusters, clearly demonstrating that they had re-differentiated in vivo. To assess whether culture expanded human ICs also secrete insulin in response to glucose, culture expanded hICs from the donors in Table A were tested per GSIS at different passages and their insulin secretion was compared with that of their parent islets (
Cell cluster formation: Islet cells from the human donors listed in Table A were tested at passages P0-P4 for their ability to form cell clusters when co-cultured with human MSCs (P3) as described in Methods. Representative images of such freshly formed cell clusters are shown in
Treatment of Mice with Human Cell Clusters
Passaged hICs in human cell clusters that were used to treat diabetic NOD/SCID mice. For in vivo testing, NOD/SCID mice were made diabetic with STZ, treated with insulin pellets (Linbits), and once stabilized, treated i.p. either with ˜2×10e5 NI/kg bw or vehicle, then followed for 7 weeks. To help assess the role of donor variability, two sets of NIs using hICs from different donors (donors 7 and 8 of Table A) and hMSCs were formed. At the beginning of the 7th week, all animals and a group of healthy controls underwent intraperitoneal Glucose Tolerance Tests (IP GTTs).
As was previously found with cNIs [2,3], hNIs dosed at ˜2×10e5 NIs per kg bw durably restore euglycemia as demonstrated by normal IP GTTs and elimination of the need for insulin in diabetic NOD/SCID mice.
Results:
Two sets of hNIs were formed, each set incorporating P3 clinical grade hMSCs and P1 islet cells from donors 7 and 8 of the research grade donors listed in Table A.
hMSCs were shown to undergo trilineage differentiation and to express MSC specific epitopes and genes. [20,21]
Passage 1 (P1) islet cells and NIs composed of islet cells from donor 7 and donor 8 and hMSCs were characterized by rtPCR for expression of islet-specific genes of interest and compared to each other as well as to fresh islets (
In
Incorporation into cell clusters of P1 ICs from either donor did not significantly affect expression of any assayed gene (see
As shown in
Once ICs were incorporated into cell clusters, Islet-specific gene expression levels from donor 7 NIs were still reduced compared to those of donor 8 NIs, but not significantly (
Also shown in
Use of hNIs in NOD/SCID mice for treatment of STZ induced Diabetes mellitus: 12 female, 13 week old NOD/SCID mice were made diabetic with one or two doses of STZ, 200 mg i.p. as described in Methods. Blood glucose levels were monitored 2× per week, and mice were considered diabetic when such levels were >300 mg/dL for 3 consecutive days, at which point, mice were treated with sub-cutaneous insulin (Linbit) pellets. Once blood glucose levels were controlled to <200 mg/dL, mice were divided into two groups of six mice each and treated i.p. either with (i) ˜2×10e5 NI/kg bw or (ii) vehicle (500 uL αMEM). For the 6 hNI treated mice, 3 were treated with NIs that incorporated donor 7's ICs, while the other 3 used donor 8's ICs. Treatment with cells from either donor offered comparable responses. As shown in
Study Design
The current, preclinical study was undertaken in anticipation of a Phase 1 Clinical Trial with two objectives: to determine (a) whether human NIs (hNIs) can also restore euglycemia, and (b) whether redosing of suboptimally controlled diabetic animals could fully restore euglycemia in streptozotocin (STC)-diabetic Non-Obese Diabetic/Severe Combined Immunodeficiency mice (NOD/SCID, Harlan), as has been previously shown for mouse cell-derived NIs (mNIs), and dog cell-derived NIs (cNIs)[3]. Since these NIs are composed of human cells, and since human MSCs do not maintain immune evasive abilities in a xenogeneic setting (unpublished results), the NOD/SCID model was used. It does reproduce, in part, the clinical situation in which recipients of allogeneic biotherapies must permanently take potent anti-rejection drugs that similarly create a life-long immune-compromised status. Passaged hICs and hNIs that were to be used to treat diabetic NOD/SCID mice were characterized for gene expression profiles by rtPCR. For in vivo testing, NOD/SCID mice were made diabetic with STZ, then randomized based on blood glucose levels into groups of 6 each. Following randomization, the mice were administered insulin pellets (Linbits, Linshin Canada) to control blood glucose levels and prevent glucotoxicity and enhance in vivo redifferentiation of the hICs within the graft. Once blood glucose levels were stabilized near normal, animals were treated i.p. either with ˜2×10e5 human cell-derived NIs/kg bw (n=6) or vehicle (n=6), then followed for 8 weeks. Once placed in a group, and until endpoint or euthanasia for humane reasons, data from all animals were included in subsequent analyses. Once blood glucose levels were determined to be no longer significantly improved compared to controls without administration of exogenous insulin, mice in each group were again treated with either 2×10e5 NIs/kg bw or vehicle, and followed for an additional 6 weeks.
Animal Model
Animal studies were conducted in adherence to the NIH Guide for the Care and Use of Laboratory Animals, and were supervised and approved by an institutional veterinarian and member of the IACUC.
Care. NOD/SCID mice were maintained in a sterile environment, and provided with sterile bedding, food and water. They were kept in a temperature and humidity controlled environment on a 12 hr light dark cycle and given free access to food and water Animal health and behavior were visually observed at least once a day during the work week, and by blood glucose and weight checks at least 2 times a week by staff, each of whom had completed CITI training in the care of rodents, and had at least 2 years' experience with mice and the procedures herein described.
Induction of diabetes and treatment. 12 female, 13 week old NOD/SCID mice were made diabetic with one to two i.p. doses of Streptozotocin (STZ; Sigma), 200 mg ip dissolved in citrate buffer (pH 4.5; Sigma), and administered under light anesthesia as described below. Tail vein blood glucose levels were monitored 2× per week, and mice were considered diabetic when such levels were >300 mg/dL for 3 consecutive days, at which point, mice were lightly anesthetized and treated with sub-cutaneous, slow-release insulin (Linbit) pellets. Once blood glucose levels were controlled to <200 mg/dL, the two groups of six mice each were lightly anesthetized, and treated i.p. either with (i) 2×10e5 human cell derived NIs (hNIs/kg b.wt.; in 500 uL DMEM (5 mM glucose) (Gibco)) or (ii) vehicle (500 uL DMEM (5 mM glucose)).
Anesthesia. Mice were anesthetized with isoflurane (Baxter), 1-5%, using an inhalation rodent anesthesia system (Euthanex). Rectal temperatures were maintained at 37° C. using a heated surgical waterbed (Euthanex).
Blood glucose and weight monitoring. Blood glucose concentrations were assessed twice per week via sterile tail vein sampling, using a 27-30 gauge needle to obtain a drop of blood, and a OneTouch Ultra 2 glucometer (level of detection, 20-600 mg glucose/dL; LifeScan). Post blood sampling, mice were observed until bleeding stopped and for a short time after for signs of tail bruising or pain (hunched appearance, head pressing, etc.). As anesthesia results in a rise of blood glucose, anesthesia was not used for blood glucose monitoring. Care was taken to minimize the pain and distress caused to mice required by handling and blood sampling for glucose monitoring, and analgesics were available as described in pain management below for any animal showing signs of pain from tail vein sampling. Animal weight was assessed twice weekly in conjunction with blood glucose monitoring.
Intraperitoneal Glucose Tolerance Tests (i.p. GTTs) and assay of human insulin. At indicated time-points post 1st and 2nd doses of hNIs, vehicle-treated and hNI-treated NOD/SCID mice, and an additional group of age-matched, non-diabetic NOD/SCID control mice (n=6) were fasted hrs, whereupon baseline blood glucose levels were measured Animals were anesthetized and 2 g glucose/kg b.wt. (dissolved in 0.5 ml serum free medium and filter sterilized; Sigma, St. Louis, MO) were administered via i.p. injection. Tail vein blood glucose levels were determined at 30 min, 60 min and 120 min post glucose administration. Human insulin levels in the sera of hNI and vehicle treated groups of mice were assayed by ELISA, following the manufacturer's instructions (Mercodia, Uppsala, Sweden). Pain management. Buprenorphine 0.05 mg/kg bw IM was available as needed for any animal appearing to suffer from pain following i.p. STZ administration, NI administration, i.p. glucose tolerance testing, or tail vein sampling.
Endpoint criteria. For all mice in this study, the following criteria were used to determine whether they should be removed from the protocol or euthanized to prevent suffering: Animals that exhibited evidence of poor health, including weight loss greater than 20%, excessive wasting (>20% compared to age/sex matched littermates), ungroomed appearance, poor activity level, labored breathing or loss of appetite/water intake, neoplasia, stupor, severe injury due to fighting with cage mates, any signs of abnormal behavior including severe aggressiveness towards handler or cage mates such as to inflict injury, lack of physical or mental alertness, or any animal appearing to be in grave distress Animals beginning to show signs of distress were monitored daily and carefully observed for general appearance, behavior and weight loss. Any animal appearing to be in grave distress or to have weight loss or muscle wasting of 20% or more were immediately euthanized to prevent further suffering.
No animal died before meeting endpoint criteria or study endpoint, but four mice in the vehicle treatment group met the criteria for euthanasia (all four exhibited excessive wasting and lack of appetite, combined with ungroomed appearance), and were euthanized on days 46 (3 mice) and 56 (1 mouse) as detailed in “Euthanasia” below, and as soon as they met those criteria.
Euthanasia. At study endpoint (applied to 8 of the 12 mice in the study), 15 weeks post first treatment with STZ, and where necessary as defined by endpoint criteria (applied to 4 mice in the vehicle treatment group) mice were euthanized using CO2 gas/4-5 L over 2-4 minutes. Death was verified by the assurance of the cessation of respiratory and cardiovascular movements by observation for at least 10 minutes.
Cells
NIs are composed of equal numbers of culture-expanded human MSCs and human Islet Cells, which spontaneously form clusters when co-cultured. Culture and NI formation are detailed below.
Islet cell culture. Research grade human islets from adult, non-diabetic donors were purchased from Prodo Labs. Islet cells were cultured by placing whole islets into tissue culture flasks and culturing them in RPMI 1640 (Life Technologies)+10% human Platelet Lysate (hPL; Cell Therapy and Regenerative Medicine, Salt Lake City, UT)+Gentamycin, Penicillin, Streptomycin (GPS; Sigma) until 90% confluent. For passaging, cells were trypsinized using 1× Trypsin EDTA (Sigma), pelleted by centrifugation at 600×g for 5 min, washed with DMEM (5 mM glucose)+10% hPL+GPS, and reseeded at a density of 2×10e5 cells into Cell Bind coated T75 flasks (Corning). Cultured Islet Cells (IC) were used at Passage 1.
MSC culture. Human, bone marrow derived MSCs were purchased from Lonza (Walkersville, MD) and cultured as previously described. MSCs were used at P3 for NI formation.
Neo-Islet (NI) formation. MSCs and Islet cells were co-cultured in DMEM (5 mM glucose)+10% hPL at a 1:1 ratio in ultra-low adhesion surface culture dishes (Corning), and NIs formed overnight as previously described.
rtPCR
Prior to in vivo administration, NIs were tested by rtPCR for expression of islet-associated genes INS, GCG, SST, PPY, PDX1, and UCN3. rtPCR was carried out as previously described, using the reagents and primers listed in. In brief, Relative Quantification, (RQ; defined as is standard as 2-MCT where CT is the Cycle Threshold), was calculated through normalization to internal (deltaCT; beta actin and beta 2 microglobulin) and external controls (delta-deltaCT; parent cells), both accomplished using the ABS 7500 Real Time PCR System and software. Results are presented as log 10(RQ)±log 10(RQmin and RQmax) so that up- and down-gene regulation is represented equally. Differences between expression levels greater than log 10(RQ) 2 or log 10(RQ)-2 were considered significant.
Statistical Analysis
Data are expressed as Mean±SEM or Mean±95% confidence interval, as indicated. Primary data were collected using Excel (Microsoft, Redmond, WA), and statistical analyses were carried out using Prism (GraphPad). Two tailed t-tests were used to assess differences between data means. A P value of <0.05 was considered significant. For rtPCR, data are presented as Log 10RQ, and statistical significance is defined as ±2.
Results
Reduced Levels of Islet-Associated Genes are Expressed in hNIs
hNIs were formed, each set incorporating P3 hMSCs and P1 islet cells from non-diabetic, adult human donors. hMSCs were obtained pre-characterized for expression of MSC-specific epitopes and genes, and for their ability to undergo trilineage differentiation (adipo-, osteo- and chondrogenic) (Lonza).
Gene expression analysis was conducted using rtPCR on the freshly formed NIs to determine their expression levels of islet endocrine genes as compared to those of whole islets. As expected from previous experiments using mouse and dog cells, and as shown in
Therapeutic Efficacy of hNIs
A single dose of hNIs improves glycemic control in diabetic mice. In order to assess the therapeutic efficacy of hNIs for the treatment of insulin-dependent DM, Diabetes was established in 12 female NOD/SCID mice, after which they were randomized into 2 groups of 6 mice each, and their blood glucose levels were controlled with slow-release insulin pellets (Linbits). Once blood glucose levels were controlled, mice were treated either with vehicle or hNIs as described in Methods. After this treatment, mice were followed for 8 weeks, at which time, an i.p GTT was conducted as described in Methods, in conjunction with an ELISA assay to detect the presence of human Insulin.
As shown in
Administration of a second dose of hNIs establishes euglycemia in previously treated dysglycemic mice. While no mice in the treated group died, 4 animals died in the vehicle treated control group, and while hNI therapy significantly improved glycemic control vs. the vehicle group for 7 weeks, normoglycemia was not maintained (
To test whether a second dose of hNIs could achieve euglycemia in the incompletely controlled mice shown in
Human insulin is detected in serum from hNI- but not vehicle-treated mice. Serum collected during the i.p. GTT depicted in
This application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/US2022/015066, filed Feb. 3, 2022, designating the United States of America and published in English as International Patent Publication WO 2022/169943 on Aug. 11, 2022, which claims the benefit under Article 8 of the Patent Cooperation Treaty to U.S. Patent Application Ser. No. 63/145,380, filed Feb. 3, 2021, the entireties of which are hereby incorporated by reference.
Filing Document | Filing Date | Country | Kind |
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PCT/US2022/015066 | 2/3/2022 | WO |
Number | Date | Country | |
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63145380 | Feb 2021 | US |