WO2015058139A1

WO2015058139A1 - Stem cell / islet cell hybrids, generation thereof, and methods for the treatment and cure of insulin-dependent diabetes mellitus

Info

Publication number: WO2015058139A1
Application number: PCT/US2014/061231
Authority: WO
Inventors: Christof Westenfelder
Original assignee: Symbiocelltech, Llc
Priority date: 2013-10-18
Filing date: 2014-10-17
Publication date: 2015-04-23

Abstract

A fused pancreatic islet cell-stem cell hybrid is disclosed, wherein the hybrid cell is capable of producing and secreting insulin. Further provided are methods for producing fused pancreatic islet cell-stem cell hybrids capable of producing insulin, including the steps of de-differentiating islet cells in-vitro, fusing de-differentiated islet cells with mesenchymal stem cells or adipose-derived stem cells, and re-differentiating the resulting hybrid cell. The resulting re-differentiated hybrid cell expresses and secretes insulin in response to glucose. Further disclosed are methods for treating a subject in need of insulin by administering an insulin-producing stem-islet cell hybrid. Further disclosed are methods for treating or reducing the severity of, e.g., insulin-dependent diabetes mellitus in a subject by administering an insulin-producing stem-islet cell hybrid to the subject, restoring physiological insulin expression, synthesis and storage, and glucose-sensitive insulin secretion.

Description

STEM CELL / ISLET CELL HYBRIDS, GENERATION THEREOF, AND METHODS FOR THE TREATMENT AND CURE OF INSULIN-DEPENDENT DIABETES MELLITUS

PRIORITY CLAIM

This application claims the benefit of the filing date of United States Provisional Patent Application Serial No. 61/893,047, filed October 18, 2013, for "HIGHLY EFFICIENT GENERATION OF INSULIN-PRODUCING STEM-BETA CELL HYBRIDS FOR THE TREATMENT AND CURE OF INSULIN-DEPENDENT DIABETES MELLITUS."

TECHNICAL FIELD

The disclosure relates generally to the field of biotechnology and cell culture and genetic engineering. The disclosure specifically relates to fused pancreatic endocrine cell - stem cell hybrids and methods of producing such cells. The disclosure also relates to the use of insulin-producing stem-islet cell hybrids for treating, e.g., insulin-dependent diabetes mellitus.

BACKGROUND

Insulin-producing β-Cells, when isolated from a donor pancreas, proliferate very poorly ex vivo, i.e., not sufficiently to generate adequate cell numbers for the treatment of insulin-dependent diabetes mellitus. In contrast, both bone marrow-derived Mesenchymal Stem Cells (MSCs) and Adipose-derived Stem Cells (ASCs)-which are undifferentiated cells-proliferate very well in cell culture. When β-Cells and other endocrine cells from the pancreatic islets are fused with MSCs or ASCs, ex vivo proliferation of these hybrid cells is modestly improved and glucose-sensitive insulin secretion by these cells remains intact both in vitro and when administered to diabetic rodents in vivo. However, as a general rule in cell biology, cells such as β-cells do not proliferate significantly while they remain fully differentiated, insulin-producing/secreting cells.

SUMMARY OF THE DISCLOSURE

Described is a fused pancreatic islet cell cell-stem cell hybrid is disclosed, wherein the hybrid cell is proliferative and capable of producing and secreting insulin.

In some embodiments, methods are disclosed for producing fused pancreatic islet cell-stem cell hybrids capable of producing insulin. In certain embodiments, methods are disclosed for de-differentiating islet cells in vitro; fusing de-differentiated islet cells with mesenchymal stem cells or adipose-derived stem cells, and re-differentiating the resulting hybrid cell. In certain embodiments, de-differentiated islet cells do not express or synthesize insulin, and lack glucose-induced insulin secretion, but proliferate very well in vitro. In certain embodiments, fusion of de-differentiated islet cells with mesenchymal stem cells or adipose-derived stem cells occurs while both cells are in the cell cycle. In certain embodiments, the resulting re-differentiated hybrid cells express and secrete insulin, c- peptide, and/or other islet hormones.

Provided are methods for treating a subject in need of insulin, the method comprising administering a fused pancreatic islet cell cell-stem cell hybrid. In embodiments, methods are provided for treating or reducing the severity of insulin-dependent diabetes mellitus in a subject, the method comprising administering a fused pancreatic islet cell-stem cell hybrid. In certain embodiments, the fused pancreatic islet cell-stem cell hybrid may be administered intraperitoneally, with restoration of physiological insulin expression, synthesis, storage, and/or glucose-sensitive insulin release.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Dissociated canine islet cells cultured for 1 day, with still largely differentiated phenotype. Proliferation is minimal.

FIG. 2. Passage 1 of de-differentiated canine islet cells (~2 weeks post initial culture), now with mesenchymal phenotype. Proliferation is excellent.

FIG. 3. Freshly isolated islets from a mouse transgenic for green fluorescent protein under the control of the insulin 1 gene promoter. Cells actively transcribing the insulin gene appear green.

FIG. 4. Islet cells from the islets shown in FIG. 3 cultured for 1 day. Cells that are actively transcribing the insulin 1 gene appear green. Cells were stained by immunohistochemistry for the presence of insulin protein (red), and for DNA (blue). Beta cells rapidly undergo de-differentiation in these culture conditions and stop actively producing insulin, though beta cells still contain previously produced and stored insulin.

FIG. 5. Graph comparing the fusion efficiency of islet cells and MSCs/ASCs from various species using IL-6 and TNF-alpha vs. PHAP in conjunction with polyethylene glycol fusion. Fusion efficiency was determined by count the fused and unfused cells (single v. 2 or more nuclei per cell) in 10 random fields of culture well 1 day post fusion. IL-6 and TNF- alpha significantly enhance fusion efficiency.

FIGs. 6A and 6B. rtPCR determined expression of Rat MSC (6A) and Rat islet cell (6B, b-cell) VCAM-1 following 6 hour, 1 day, 2 day, or no exposure to IL-6 and TNF- , normalized to values for no exposure. VCAM-1 is highly up-regulated in MSCs and modestly up-regulated in islet cells following exposure to these inflammatory cytokines.

FIG. 7. Canine fused (P2 abdominal ASCs + PI dedifferentiated islet cells) cells that had been subjected to redifferentiation, then passaged into normal medium, and stained for EdU (red) and insulin (green). Passaging into normal medium promotes the E to M transition of the single nucleated cells present in the culture. These take up EdU as they begin to divide. The multinucleated cells (see arrows) very rarely take up EdU.

FIGs. 8A and 8B. Gene expression profiles of mouse (8A) and dog (8B) redifferentiated hybrid cells demonstrate expression of islet cell specific genes.

FIG. 9. Glucose stimulated release of insulin (ELISA) from 50 freshly isolated dog islets from 1 hour exposure to medium containing 2.8 mM glucose and 1 hour exposure to medium containing 14 mM glucose.

FIG. 10. Glucose stimulated release of insulin (ELISA) from 2x10⁵ dog islet cells post 1 hour exposure to medium containing 2.8 mM glucose (blue bars) and 1 hour exposure to medium containing 14 mM glucose (red bars) after culture for 1 day (dl) and 4 days (d4).

FIG. 11. Glucose stimulated release of insulin (ELISA) in cultured dog islet cells prior to step 2 redifferentiation (Fuse before RDM and Fuse no RDM), one week after culture in step 2 redifferentiation medium (fusion RDM 1 week and Fusion no RDM 1 week) and after culture in step 2 redifferentiation medium for 2 weeks.

FIG. 12. A culture of fused canine β cells and MSCs stained for the three primary islet hormones, insulin (green), glucagon (red; double headed arrow) and somatostatin (purple; arrow), and nuclei (blue). This image demonstrates that the generated hybrid cells reproduce the cell biology of an intact islet in the physiological ratios.

FIGs. 13A and 13B. Rats made diabetic and treated allogeneically with hybrid cells show restoration of normalized blood glucose levels within a week following treatment (13 A). This restoration persists, and treated animals have normal intraperitoneal glucose tolerance tests at end of study (13B). MODE(S) FOR CARRYING OUT THE INVENTION The disclosed methods and cells overcome the limited ability to generate sufficient therapeutic doses of isolated and cultured islet cells. The islet cells may, e.g., be isolated from a single pancreas donor. Islet cells include, e.g., alpha cells, beta cells, delta cells, PP cells (also known as gamma cells) and epsilon cells.

Described is a fused pancreatic islet cell-stem cell hybrid. The hybrid cell may express insulin, glucagon, somatostatin or other hormones. The hybrid cell may secrete insulin, c- peptide, and/or amylin when exposed to glucose. The hybrid cells may have more than one nucleus. For example, a hybrid cell may comprise at least one pancreatic islet cell nucleus and at least one stem cell nucleus.

Methods are disclosed for generating islet cell/stem cell hybrids that comprise:

de-differentiating islet cells in vitro in order to facilitate their proliferation;

fusing de-differentiated islet cells with stem cells, to generate hybrid cells; and treating the hybrid cells to facilitate re-differentiation in order to reestablish pancreatic hormone production.

Differentiated islet cells express, e.g., insulin, but do not proliferate, or proliferate only minimally, in vitro. Isolated islet cells may be induced to de-differentiate in vitro. As used herein "de-differentiated" islet cells or islet cell nuclei are cells or nuclei that no longer express or produce insulin when challenged with glucose. The process of de-differentiation is also referred to herein as an Epithelial-Mesenchymal transition or an "E to M" transition. Dedifferentiated islet cells may proliferate in culture at a rate superior to differentiated islet cells. De-differentiation of the islet cells may immediately silence insulin expression, insulin synthesis, insulin storage, and/or glucose-induced insulin secretion in these cells. Isolated islet cells may be from any suitable host {e.g., rodent, canine, human, or other mammal).

De-differentiated islet cells may be allowed to proliferate in vitro to form a large pool of cells that fuse very well with stem cells.

De-differentiation may be achieved by culturing islet cells in a de-differentiation medium. De-differentiation 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 de-differentiation 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, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28 29, or 30 nM.

Culturing of isolated islet cells on laminins (e.g., laminin-41 1 and 51 1), and addition of suitable media, may improve cell adhesion in culture, support cell survival, and moderately boost proliferation. For de-differentiation, islet cells may be plated on a suitable substrate that allows for attachment. In specific embodiments, the substrate may include Laminin-41 1 and/or Laminin-51 1. In more a specific embodiment, β-cells may be plated on tissue culture flasks or wells coated with Laminin-41 1 and/or Laminin-51 1 and placed in RPMI or other suitable culture medium and supplemented with 10%-20% fetal bovine serum or other serum, and glutamine/penicillin/streptomycin. The culture medium may also be supplemented with at least 10 nM Exendin-4.

The pancreatic islet cells and/or de-differentiated islet cells may be fused to a stem cell to create a hybrid. Examples of stems cells to which pancreatic islet cells may be fused include, but are not limited to, mesenchymal stem cells (MSCs) and adipose-derived stem cells (ASCs). MSCs and ASCs are undifferentiated stem cells that proliferate well, and do not produce insulin. In embodiments, stem cells may be fused with de-differentiated islet cells to produce fused pancreatic islet cell cell-stem cell hybrids. This may be accomplished while both cells (fusion partners) are in the cell cycle. The cells may be fused using any cell fusion protocol, e.g., electrofusion or polyethylene glycol (PEG).

In certain embodiments, de-differentiated islet cells may be fused with MSCs and/or

ASCs. For fusion, the de-differentiated islet cells may be co-cultured overnight with MSCs and/or ASCs or the de-differentiated islet cells may be layered over established MSCs and/or ASCs.

In certain embodiments, the islet cells and/or the stem cells may be pretreated or conditioned prior to fusion. For example, the islet cells and/or the stem cells may be exposed to TNF-alpha and/or IL-6 in order to enhance fusion efficiency. The exposure to TNF-alpha and/or IL-6 maybe overnight, at least 2 hours, or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 25, 26, 27, or 28 hours. For conditioning, the TNF- alpha may be present in the culture medium at a concentration from 0.1 to 100 ng/ml, from 1 to 50 ng/ml, or at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28 29, or 30 ng/ml. For conditioning, the IL-6 may be present in the culture medium at a concentration from 0.1 to 100 ng/ml, from 1 to 50 ng/ml, or at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28 29, or 30 ng/ml. Alternatively, cell may be pretreated with phytohemagglutinin (PHAP) to enhance fusion efficiency.

Pretreatment of mouse MSC + islet cells with IL-6 + TNF-alpha prior to fusion has been shown to enhance fusion efficiency by 18-58%, as compared to when these agents are absent. Compare Table 1 and Table 2, and see FIG. 5.

# fused in # unfused

Field field in field Total % Fused

1 25 22 47 53%

2 34 23 57 60%

3 27 15 42 64%

4 29 23 52 56%

5 30 15 45 67%

6 16 26 42 38%

7 34 14 48 71%

8 18 21 39 46%

9 20 16 36 56%

10 27 16 43 63% ave 26 19.1 45.1 57%

Table 1 : Fusion efficiency of mouse MSC + islet cells pretreated with TNF-alpha and IL- 6 (average of 10 counted fields).

# fused in # unfused in

Field field field Total % Fused

1 2 18 20 10%

2 2 17 19 11%

3 2 14 16 13%

4 3 25 28 11%

5 9 24 33 27%

6 4 16 20 20%

7 12 22 34 35%

8 2 17 19 11%

9 7 15 22 32%

10 2 20 22 9% ave 4.5 18.8 23.3 18% Table 2: Fusion efficiency of mouse MSC + islet cells not pre-treated with TNF-alpha and IL-6 (controls). Pre-fusion preconditioning of MSCs, ASCs and islet cells with IL-6 and TNF-a results in up regulation of cell adhesion molecule VCAM-1 in all cells, and no major change in the expression of VCAM-l 's cognate adhesion partner Integrin a4 βΐ . This results in enhanced adhesion of the respective cell fusion partners and greatly enhanced fusion efficiency (see FIG. 5).

De-differentiated islet cells proliferate well, but do not express or secrete insulin. In some embodiments, de-differentiated islet cells are allowed to proliferate to generate sufficient numbers for subsequent manipulation. In certain embodiments, once sufficient dedifferentiated islet cells have been generated and fused to form hybrid cells, the hybrid cells are treated with an islet cell or beta cell-specific re-differentiation medium. Re-differentiation of the hybrid cells restores insulin production, resulting in the re-expression of physiological insulin expression, synthesis, storage and glucose-sensitive insulin release. In embodiments, redifferentiated hybrid cells maintain multiple nuclei. For example, re-differentiated fused islet cell-stem cell hybrids may contain at least one re-differentiated islet cell nucleus and at least one stem cell nucleus, such as an MSC nucleus or an ASC nucleus.

Described is the redifferentiation of a fused de-differentiated islet cell-stem cell hybrid to generate a redifferentiated hybrid cell. Redifferentiation, as used herein, refers to the treatment of fused de-differentiated islet cell-stem cell hybrids to generate a redifferentiated hybrid 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 fused de-differentiated islet cell-stem cell hybrid 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 fused de-differentiated islet cell-stem cell hybrid in the culture medium containing a low level of glucose for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 -14, 2-13, 3-12, 4-10, or 5-9 days.

In a second step, the fused de-differentiated islet cell-stem cell hybrid 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, 1 1, 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, 1 1, 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 uM. The triiodothyronine may be present in the culture medium at a concentration from 0.1 to 100 uM. The GLP-1 receptor agonist may be Exendin-4. The second step may include culturing the fused de-differentiated islet cell-stem cell hybrid 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, 10-28, 1 1- 27, 12-26, 13-25, or 14-29 days.

Methods are also disclosed for generating insulin-producing stem-islet cell hybrids, the methods comprising:

de-differentiating islet cells in vitro, wherein the de-differentiated islet cell does not express, store, or synthesize insulin, and does not secrete insulin in response to glucose;

fusing de-differentiated islet cells with mesenchymal stem cells or adipose-derived stem cells, to generate hybrid cells, wherein both cell fusion partners are in the cell cycle, and wherein the hybrid cells show no insulin expression or secretion; and

treating the hybrid cells to facilitate re-differentiation, wherein the re-differentiated hybrid cells show physiological insulin expression, synthesis, storage and glucose-sensitive insulin release.

In some embodiments, a method is provided for generating insulin-producing fused islet cell-stem cell hybrids through a substantial expansion in the amount of starting material (dedifferentiated islet cells) for subsequent fusion with proliferating MSCs or ASCs.

Also described is a method of treating a subject in need of insulin, using redifferentiated hybrid cells, is disclosed. In some embodiments, redifferentiated hybrid cells are administered intraperitoneally into the subject. In certain embodiments, administration of the redifferentiated hybrid cells to the subject restores insulin production, secretion, and glucose-responsiveness.

Further described is a method of treating or reducing the severity of insulin-dependent diabetes mellitus in a subject by redifferentiated hybrid cells is disclosed. In some embodiments, redifferentiated hybrid cells are administered intraperitoneally into the subject. In certain embodiments, administration of the redifferentiated hybrid cells to the subject results in the cure of insulin-dependent diabetes mellitus.

EXAMPLES

The following examples are provided for illustrative purposes only and are not to be construed as limiting.

Example 1: Culture Media used for Experiments

Prior to fusion, cells are cultured as follows.

Rodent:

Islet cells are grown in DMEM/F12, RPMI, or alpha MEM + 20% FBS + GPS (glutamate, penicillin, streptomycin), and on Laminin-511 coated plates for P0.

MSCs and ASCs are grown in DMEM/F12, RPMI, or alpha MEM + 10% FBS + GPS.

Canine:

Islet cells are grown in DMEM/F12 (RPMI or alpha MEM) + 20% dog serum + GPS, and on Laminin-511 coated plates (flasks, wells, etc.) for P0.

MSCs and ASCs are grown in DMEM/F12 (RPMI or alpha MEM) + 10% dog serum + GPS.

Human:

Islet cells are grown in PIMS + PIM(G) + PIM(ABS), all from ProdoLabs (Irvine, CA), and on Laminin-511 for P0. For subsequent passages, Islet cells are grown in DMEM/F12 (or alpha MEM) + 20% FBS (or platelet lysate) + GPS.

MSCs and ASCs are grown in DMEM/F12 (or alpha MEM*) + 10% FBS (or 3-5% human platelet lysate) +GPS. For Electroiusion, cells are washed 3x in Cytofusion medium (BTX) prior to final suspension in Cytofusion medium and fusion.

PEG fusion media and buffers are as described elsewhere, and are 50% Polyethylene Glycol 1500 from Roche, and PHAP from Sigma.

Redifferentiation medium

Step 1 :

DMEM containing 5.6 mM D-Glucose

1% FBS (or dog serum or platelet lysate, depending on the cells' species)

ITS

Pen/Strep

Step 2:

DMEM containing 25 mM D-Glucose

1% FBS (or dog serum or platelet lysate, depending on the cells' species)

ITS

Pen/Strep

N2 supplement

B27 supplement

10 mM Nicotinamide

lO mM Exendin 4

2 mM Activin A

10 μΜ Alk-5 inhibitor II

1 μΜ Triiodothyronine.

Example 2: De-differentiation of Islet Cells

Islet cells are either (1) dissociated with trypsin and cells plated in Laminin-51 1 and/or Laminin-411 (20 μg/ml) precoated Tissue Culture (TC) wells or flasks, or (2) whole islets are plated in Laminin-511 and/or Laminin-41 1 coated TC wells. See FIG. 1. In both cases, cells are cultured and allowed to propagate in RPMI or other suitable growth medium supplemented with 20% Fetal Bovine Serum (FBS) + glutamine/penicillin/streptomycin (GPS) + Exendin 4 (10 nM for rodent cell cultures) until confluence (all supplements are commercially available). This process takes ~ 1-2 weeks, islet cells become dedifferentiated within a matter of days, judging from immunohistochemistry (1HC) for insulin presence, Insulin Enzyme Linked Immunosorbent Assay (ELISA), and from murine cell lines transgenic for Green Fluorescent Protein (gfp) under the control of the insulin 1 gene promoter. See FIGS. 2-4.

Example 3: Determining whether dedifferentiation of islet cells overcomes the problem of limited numbers of islet cells.

In order to determine whether islet cells could be cultured, dedifferentiated, propagated and passaged, thus allowing the generation of enough cells for a therapeutic hybrid product, mouse, rat, and canine islets were isolated, and human islets were purchased from commercial vendors. The islets were either enzymatically dissociated and plated, or plated whole on Laminin-511 coated dishes and subjected to de-differentiation. In all cases, the dedifferentiated islet cells propagated well at P0. Acinar cells did not attach. Immunohistochemistry and other methods demonstrated that the islet cells from all tested species underwent an Epithelial to Mesenchymal transition (de-differentiation).

Mouse: Mouse islet cells can be cultured, and will undergo an Epithelial to Mesenchymal transition (de-differentiation).

Rat: As for mice above, rat islet cells can be cultured and will undergo Epithelial to Mesenchymal transition (de-differentiation).

Dog: De-differentiated dog islet cells can be passaged multiple times.

Human: De-differentiated human islet cells can be passaged multiple times.

In summary, de-differentiation does allow expansion and overcomes the lack of starting material. Example 4: Fusion of MSC and/or ASC with De-differentiated Islet Cells

PEG based fusion

MSC and/or ASC cells are fused with de-differentiated islet cells by either co-culture of cells overnight or layering of cells overnight (islet cells on top of established MSC or ASC cultures) followed by treatment with PHA-P (100 pg/ml) for 30 minutes. This treatment is followed by fusion with polyethylene glycol (PEG; 50%) for 1 minute. Post-fusion, culture medium is changed to normal growth medium (DMEM-F12, 10% FBS + GPS) until cells are confluent or sub-confluent.

Electrofusion

Electrofusion is accomplished by mixing high densities of cultured MCS and/or ASCs and de-differentiated islet cells, in cytofusion medium, placing the cells in a fusion chamber, and subjecting the cells to a long AC pulse to align the cells, followed by several short DC pulses and a recovery period.

In order to test the fusion efficiency of PEG vs. Electrofusion, dog ASCs and dog islet cells were stained green and red, respectively, mixed at a ratios of 1 :1 , 1 :2 and 2:1, and cells from each group were further subdivided into additional 4 groups: (a) mixed control for PEG (unfused); (b) PEG fused; (c) mixed control for Electrofusion (unfused); (d) electrofused. Control cells were subjected to the same steps as their fused counterparts with omission of the fusion step. 24 hours post-fusion, an aliquot of cells from each group was assessed for viability, and a second aliquot was reserved for flow cytometry analysis (FACS). Cells were then fixed, stained with DAPI, and imaged. Imaged cells were analyzed for percent fused cells (red-green) and number of nuclei per cells. PEG fusion efficiency (10-50%) was consistently and significantly more efficient than Electrofusion (8-22%), no matter the ratio of cells tested.

Islet cell gene expression profiles for redifferentiated PEG and Electrofused cells were also compared. Electrofusion followed by redifferentiation gave better beta cell-like gene expression profiles.

. Electrofusion: Electrofusion is preferred for our applications due to

Scalability

Lack of toxins in the fusion process Better gene expression of islet-related genes (in mouse: pdx-1, glut-1, glut-2, pax6, nkx6.1, and somatostatin. In dog: Insulin, glucagon, somatostatin, pdxl, pax4, nkx6.1, and pax6; see Example 9 below). Ratios of Cell Fusion Partners: 1 :1 or 1 :2 Islet to ASC and/or MSC, work equally well for generating islet related gene expression.

Example 5: Preconditioning MSCs/ASCs and islet cells with IL-6 and TNF-alpha boosts fusion efficiency

Zhang, et al. (Circ Res 2007, 100:693-702), the contents of which are incorporated herein by this reference, found IL-6 + TNF-alpha + hypoxia increased spontaneous fusion of cardiomyocytes and HPCs, and that these factors stimulate cell fusion through up-regulation of VCAM-1 and alpha4 and betal integrin genes.

Fusion using IL-6 and TNF-alpha is accomplished by layering or co-culturing dedifferentiated islet cells, stained red, with passaged ASCs or MSCs, stained green, in the presence of IL-6+TNF-a (10 ng/ml each) overnight, followed by fusion with PEG (50%, for 1 minute), and recovery overnight in DMEM-F12 + 1% FBS. Fusion using PHAP + PEG is accomplished as indicated In Example 2.

Fusion efficiency is determined by counting the fused and unfused cells (single vs. >2 nuclei per double colored cell) in 10 random fields of a culture well 1 day post fusion. IL6 and TNF- alpha significantly enhanced fusion efficiency. The results are presented in FIG. 5.

Fusion of mouse cells: freshly isolated mouse islets from GFP+ C57BL/6 mice were trypsinized to single cells, and layered over cultured mouse MSCs (-2:1 ratio). After 24 hours, the co-cultured cells were treated with IL6 and TNF-alpha overnight, or with PHAP for 30 minutes and fused with PEG for 1 minute. After fusion and overnight culture in DMEM F12 +10% FBS, 10 fields from each condition were counted for fused vs. unfused cells (see FIG. 5).

Fusion of Human 1 cells: PEG fusion was carried out as above between P3 MSCs and dedifferentiated, P3 islet cultured islet cells. Cells were co-cultured at a 1 :2 (MSC to Islet) ratio (see FIG. 5).

Fusion of Human 2 cells: PEG fusion was carried out as above between either P3 MSCs and dedifferentiated, P3 cultured islet cells at a 1 :2 ratio (MSCs to Islet cell), or P4 MSCs and dedifferentiated P2 cultured islet cells at a 1 :4 ratio (MSCs to islet cells), as indicated on the x-axis legend (see FIG. 5).

Fusion of Canine cells: PEG fusion was carried out as above between PI ASCs and PI dedifferentiated cultured islet cells at a 1 :2 ratio (ASCs to islet cells) (see FIG. 5).

To assess a mechanism by which IL-6 and TNF-a exposure might enhance fusion efficiency, VCAM-1, 4 and βΐ integrin subunits, and glplr gene expression at various time points post exposure were examined by rtPCR. In summary: (A) IL-6 and TNF-a exposure induce strong up-regulation of VCAM-1 in MSCs, which begins by 6 hours post exposure, and continues for at least 48 hours (FIG. 6A); (B) a4 and βΐ are only modestly up-regulated at 6 hours, but down-regulated subsequently; (C) VCAM-1 is modestly up-regulated in islet cells by 24 hours, but this is not sustained by 48 hours (FIG. 6B). (D) a4 and βΐ appear to be down-regulated at both 24 and 48 hours post exposure. (E) Glplr was undetected in both MSCs and islet cells post exposure.

We have found that replacing PHAP with IL-6 and TNF-alpha promotes polyethylene glycol mediated fusion of de-differentiated islet cells with ASCs and/or MSCs. This observation holds for mouse, rat, and human cells, but not for dog cells (see FIG. 5). In contrast to Zhang's findings, exposing either MSCs or islet cells or both to hypoxia did not affect fusion efficiency. Example 6: Lack of Proliferation of Hybrid Cells

Hybrid cells that are generated from the fusion of de-differentiated islet cells and MSCs and/or ASCs (polykaryons) do not proliferate in vitro. FIG. 7 depicts canine fused cells that have been subjected to redifferentiation, then passaged into normal medium, and stained for 5-ethynyl-2'-deoxyuridine (EdU; red). Passaging back into normal medium again promotes the de-differentiation of the single nucleated cells present in the culture. These take up EdU as they begin to divide (red). The multinucleated cells (see arrows) very rarely take up EdU, indicating that hybrid cells do not divide. This result has been confirmed in rodent cells. Example 7: Purification of hybrid cells

Purification of hybrid cells is accomplished by one of several methods including:

Heparin, Protamine-S04, Ferumoxitol labeling;

FACS; Cell surface antibody labeling (magnetic beads attached to antibodies) and chromatography; and

Other methods known to those familiar with the art Collectively, these methods result in 92 to 94% purification of fused/labeled cells with a recovery of viable cells, depending on the purification method used, of between 2% and 80%.

Example 8: Redifferentiation of Fused De-Differentiated Islet Cell-Stem Cell Hybrids

Re-differentiation of fused de-differentiated islet cell-stem cell hybrids is achieved using commercially available additives, in a two-step process:

Step 1: grow fused de-differentiated islet cell-stem cell hybrids for 5-9 days in serum free DMEM containing 5.6 mM D-glucose and supplemented with:

1% FBS (or dog serum or human platelet lysate, depending on the cells' species); - ITS; and

Pen/Strep.

Step 2: After 5-9 days, switch to Redifferentiation Medium (RDM) and culture for 14 to 21 additional days. RDM is DMEM containing 25 mM D-glucose and supplemented with:

- 1% FBS (or dog serum or platelet lysate, depending on the cells' species);

- ITS;

Pen/Strep;

N2 supplement;

B27 supplement;

- 10 mM Nicotinamide;

10 mM Exendin 4; and

- 2 mM Activin A

- 10 uM Alk-5 inhibitor II

1 μΜ Triiodothyronine. Example 9: Gene expression profiles of redifferentiated hybrid cells

To determine whether redifferentiated hybrid cells express islet genes, hybrid mouse and dog cells were cultured in RDM (2 steps) for 3 weeks, and assessed by PCR for the expression of various islet genes, including pdx-1 , glutl, glut2, pax 4, pax6, nkx6.1, insulin, glucagon and somatostatin. Fused de-differentiated islet cell-stem cell hybrids cutured in redifferentiation in RDM for 3 weeks results in redifferentiated hybrid cells that express pdx-

I, glutl, glut2, pax 4, pax6, nkx6.1 , insulin, glucagon and somatostatin (see FIG. 8).

Example 10: Glucose Stimulated Insulin Secretion in redifferentiated hybrid cells

FIG. 9 shows glucose stimulated release of insulin (via ELISA) from 50 freshly isolated dog islets from 1 hour exposure to medium containing 2.8 mM glucose and 1 hour exposure to medium containing 14 mM glucose. FIG. 10 shows Glucose Stimulated Release of Insulin (ELISA) from 2x10⁵ dog islet cells post 1 hour exposure to medium containing 2.8 mM glucose (blue bars) and 1 hour exposure to medium containing 14 mM glucose (red bars) after culture for 1 day (dl) and 4 days (d4), demonstrating rapid dedifferentiation of the islet cells.

To determine whether redifferentiated hybrid cells could release insulin in response to glucose, media from redifferentiated cells exposed to 2.8 and 14 mM glucose were tested for insulin secretion by ELISA at various times post redifferentiation. Results are shown in FIG.

I I , and clearly demonstrate that redifferentiated hybrid cell show controlled release of insulin in response to glucose.

Example 11: Immunohistochemistry for islet hormones in redifferentiated hybrid cells

Redifferentiated dog hybrid cells were fixed and examined by immunohistochemistry in vitro for the presence of insulin (green), glucagon (red), and somatostatin (purple) proteins. As seen in FIG. 12, such cultured cells express all three hormones, predominantly insulin and glucagon.

Example 12: Allogeneic treatment of streptozotocin induced diabetes in Rats with hybrid cells.

In order to test whether hybrid cells could reverse hyperglycemia in diabetic animals, male Sprague Dawley rats with mild streptozotocin (STZ)-induced diabetes (50 mg/kg bw in citrate buffer I/P) were treated with 2xl 0⁶ cells/kg bw injected intraperitoneally. The injected cells were from frozen stocks of cultured hybrid cells derived from Fischer 344 rats. Treatment was administered under anesthesia 7 days post-STZ induction. Negative controls were rats injected with citrate buffer alone. Positive controls, where shown, are untreated STZ-induced diabetic rats. As can be seen in FIGs. 13A and 13B, rats made diabetic and treated in an allogeneic protocol with hybrid cells show sustained restoration of normoglycemia and a normalized intraperitoneal glucose tolerance test.

While the application has been described with certain embodiments and examples, it can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of de-differentiating a pancreatic islet cell in vitro, the method comprising:

culturing trypsin dissociated islet cells; or

culturing whole islets.

2. The method according to claim 1, wherein the pancreatic islet cell is plated on a laminin.

3. The method according to claim 1, wherein the laminin is laminin-411 and/or laminin-51 1.

4. The method according to claim 1 , wherein the de-differentiated pancreatic islet cell proliferates in culture.

5. The method according to claim 1, wherein the pancreatic islet cell is isolated from a mammal, canine, rodent, or human.

6. A de-differentiated pancreatic islet cell produced by the method according to any of claims 1-5.

7. A method of generating a fused de-differentiated pancreatic islet cell/stem cell hybrid, the method comprising:

fusing the de-differentiated pancreatic islet cell of claim 6 with a stem cell to generate a hybrid cell.

8. The method according to claim 7, the method further comprising:

conditioning the de-differentiated pancreatic islet cell with TNF-alpha and IL-6 prior to fusion.

9. The method according to claim 8, wherein the stem cell is conditioned with TNF-alpha and IL-6.

10. The method according to claim 8, wherein conditioning the pancreatic islet cell with TNF-alpha and IL-6 comprises exposing the pancreatic islet cell to at least 10 ng/ml

TNF-alpha and at least 10 ng/ml IL-6 for at least 1 hours.

1 1. The method according to claim 7, wherein the stem cell is a bone marrow- derived mesenchymal stem cell or an adipose-derived stem cell.

12. The method according to claim 7, wherein the hybrid cell lacks glucose- induced insulin secretion.

13. The method according to claim 7, wherein the fusion occurs while the de- differentiated islet cell and the stem cell are in the cell cycle.

14. A de-differentiated pancreatic islet cell/stem cell hybrid produced by the method according to any of claims 7-13.

15. The de-differentiated pancreatic islet cell/stem cell hybrid of claim 14, wherein the hybrid cell does not express insulin.

16. The de-differentiated pancreatic islet cell/stem cell hybrid of claim 14, wherein the hybrid cell comprises more than one nucleus.

17. The de-differentiated pancreatic islet cell/stem cell hybrid of claim 16, wherein the hybrid cell comprises at least one pancreatic islet cell nucleus and at least one stem cell nucleus.

18. A method of generating an insulin-producing stem cell-islet cell hybrid, the method comprising:

treating the hybrid cell of any one of claims 14 to 17 to facilitate re-differentiation.

19. The method according to claim 18, wherein the insulin-producing stem cell- islet cell hybrid does not proliferate but shows insulin expression and secretion.

20. The method according to claim 18, wherein re-differentiation is facilitated utilizing a beta-cell specific re-differentiation medium.

21. A re-differentiated hybrid cell produced by the method according to any of claims 18-20.

22. The re-differentiated hybrid cell of claim 21, wherein the hybrid cell expresses and secretes insulin when exposed to glucose.

23. The re-differentiated hybrid cell of claim 21, wherein the hybrid cell comprises more than one nucleus.

24. The re-differentiated hybrid cell of claim 23, wherein the hybrid cell comprises at least one pancreatic islet cell nucleus and at least one stem cell nucleus.

25. The re-differentiated hybrid cell of any of claims 21-24 for use in a subject in need of insulin therapy.

26. The re-differentiated hybrid cell of any of claims 21-24 for use in intraperitoneal injection use in a subject in need of insulin therapy.

27. A re-differentiated hybrid cell of any of claims 21-24 for use in a method of treating or reducing the severity of insulin-dependent diabetes mellitus in a subject.