PROLIFERATED ISLETS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of priority to U.S. Provisional Application Serial No. 60/826,947, filed September 26, 2007, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention:

[0003] The present invention relates to proliferated islet cells (PI), which are useful in islet cell transplantation.

[0004] 2. General Background and State of the Art:

[0005] At present, the biggest problems concerning islet transplantation (IT) are the short supply of islets and immune rejection of grafts. The field of islet transplantation involves a gathering of the most difficult skills from different fields of science.

Therefore, in order to succeed, perfect coordination and execution of islet transplantation by highly trained professionals is a must. But all this can end in failure because of immune rejection, and the shortage of islet supply will obstruct the wide usage and further development of this technology.

[0006] By way of background, Dr. James Shapiro in Canada developed a new immunosuppressor regimen that greatly suppresses the immune rejection, significantly improving the success rate of IT. Combining this and conventional IT methods, the average life of the grafts is 3 years, and as a result, the patient returns to insulin dependence after this period. Even this is unsatisfactory. Therefore, new methods are required to overcome the problems associated with the short supply of viable islet cells and immune rejection.

SUMMARY OF THE INVENTION

[0007] The present invention is directed to an isolated proliferated islet cell which produces a protein that is not produced in fresh islet cell. The protein may be reverse

transcriptase (RT), ornithine decarboxylase (ODC), protein disulfide isomerase (PDl), C-

MYC, SmCX or Acyl-CoA. The amount of the protein may be increased by about 1.25 fold to about 3 fold compared with fresh islet cell. The protein may be increased by about

1.5 fold to about 2.5 fold compared with fresh islet cell, or the increase may be about

1.75 fold to about 2.25 fold, or the increase may be about 2 fold. The protein may be produced recombinantly.

[0008] In another aspect, the invention is directed to a proliferated islet cell which does not produce proteins that are produced in fresh islet cell. In this instance, the protein may be an albumin precursor, beta actin, cytokeratinδ or PlO binding protein.

[0009] The proliferated islet cell may also produce an increased amount of a stress resistance protein compared with fresh islet. The stress resistance protein may be an oxidative stress resistance protein, such as glutathione peroxidase (GPX-I), an anti- apoptotic protein, or catalase.

[0010] In another aspect, the invention is directed to a method of regenerating pancreatic beta cells in a patient comprising transplanting the proliferated islet cells according to any of the above to the patient. In particular, the generated protein may be

ODC, PDI, Acyl-CoA, or C-MYC.

[0011] In still another aspect, the invention is directed to a method of regenerating pancreatic beta cells, comprising transplanting the proliferated islet cell according to any of the above types to the pancreas of a patient.

[0012] In yet another aspect, the invention is directed to a method of regenerating pancreatic beta cells, comprising making an islet cell that recombinantly expresses a protein that is not produced in fresh islet cell, and transplanting the islet cell that recombinantly expresses the protein to a patient in need thereof. The protein may be

ODC, PDI, Acyl-CoA, C-MYC.

[0013] In another aspect, the invention is directed to a method of regenerating pancreatic beta cells, comprising making an islet cell that recombinantly expresses an oxidative stress resistance protein, and transplanting the islet cell that recombinantly expresses the protein to a patient in need thereof. The oxidative stress resistance protein may be glutathione peroxidase (GPX-I).

[0014] These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;

[0016] FIGURE 1 shows two-dimensional gel of proteins that are produced in fresh islets (FI). Circled spots are proteins expressed only in FI, and not proliferated islets (PI).

[0017] FIGURE 2 shows two-dimensional gel of proteins that are produced in proliferated cells (PI). Circled spots are proteins expressed only in PI, and not in fresh islets (FI).

[0018] FIGURES 3 shows stress gene expression levels in fresh islets (FI) and proliferated cells (PI).

[0019] FIGURES 4A and 4B show examination of transplanted islets with light microscopy. Insulin-secreting β-cells are detected by insulin staining (red). 4A. Fresh islets transplanted into spleen. 4B. Proliferated islets transplanted into spleen.

[0020] FIGURES 5A and 5B show examination of transplanted islets with electron microscopy. Transplanted islets are imaged through electron microscopy. 5A. Structure of transplanted fresh islet: growth of fibrotic tissue, destroying islets; high polysome increase. 5B. Structure of transplanted proliferated islet: normal structure without increase of fibrotic tissue and polysome.

[0021] FIGURE 6 shows pancreatic islet EQ number of mice transplanted with PI or

FI. In mice transplanted with FI, the number of pancreatic islets did not increase as the period of normoglycemia prolonged. Mice transplanted with PI showed increasing islet

EQ number in proportion with the duration of normoglycemia achieved.

[0022] FIGURE 7 shows blood glucose level changes after removal of PI or Fl.

[0023] FIGURE 8 shows blood glucose level changes after removal of PI or FI.

[0024] FIGURES 9A-9C show islet regeneration. 9A-9B show islet regeneration

(especially β-cell regeneration). Mouse pancreatic islets after proliferated islet

transplantation (PIT) are shown. 9C shows islet neogenesis. New born islets were detected from mouse that maintained normoglycemia 6 months after PIT (300 IEQ). [0025] FIGURES 10A-10B show morphology of A. fresh islet; and B. proliferated islet using electron microcroscopy.

[0026] FIGURES 1 IA-I I B show morphology of A. fresh islet; and B. proliferated islet using electron microcroscopy.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In the present application, "a" and "an" are used to refer to both single and a plurality of objects.

[0028] To overcome the shortage of islet supply, the inventor has been able to proliferate islets in vitro. When native pancreatic islets and proliferated islets (PI) are compared under light microscope, they appear to be structurally identical. However, there are significant differences at the molecular, biochemical and functional levels between these cell types.

[0029] In the present application several proteins are named as being expressed in either FI or PI or both. It is to be understood that the so-named protein is not limited to a particular sequence of the protein, and may encompass homologs, isoforms and mutants of the protein, and may generally include proteins belonging to a biochemically categorized family of the named proteins.

[0030] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.

EXAMPLES

[0031] EXAMPLE 1 - Proliferation of islet

[0032] The islet is proliferated immediately after extracting from mice. It is imperative that plasma extracted from the mice be added. Manufacture of the plasma

starts from putting 2~5mL of blood extracted from the mice into a 15mL Falcon Tube (Product No. 352097, Becton Dickinson, USA) and leaving it at room temperature for 2~4 hours. It is then put in a centrifugal separator at 300Og for 10 minutes after which the 1.5~2mL supernatant is put in a tube. It is then kept at 4 C for one night and put in a centrifugal separator at 3000g for 10 minutes after which the supernatant is kept and used at -20 C. First, a DMEM culture medium [1 mg pyruvate (100 μ 1) (Cat. No. 1 1360-070, GlBCOBRL, USA), 0.25 μ g (100 μ 1) hydrocortisone (H-0135, Sigma USA), lOOunit/lOO u g (I mI) penicillin/streptomycin (Cat. No. 15140, GIBCO, USA), 4,456mg (4 μ 1) β-mercaptoethanol (Product No. M-6250, Sigma, USA), 14.6mg (500 μ 1) L- glutamate (Cat. No. 25930-081 , GlBCO, USA), 283.3mg Hepes buffer (Cat. No. 1560- 080, GIBCO, USA)] is used to make the basic culture solution. 6mL of this solution is mixed with 600 μ 1 of blood plasma along with radical scavenger Nicotinamide (N-0636, Sigma, USA) 61mg, ITS (insulin transferring selenite) (1-3769, 1-2139, Sigma, USA), anti-necrosis and anti-apoptosis IGF-I, 2(1-3769, 1-3239, Sigma, USA) at concentrations of 5 μ g and 600ng respectively, VEGF (vascular endothelial growth factor) (E-1264, Sigma, USA) 12ng, caspase inhibitory EGF (E-1364, Sigma, USA) 60ng, NGF(N-6009, Sigma, USA) 20ng, linoleic acid-BSA (Linoleic acid-BSA, L-8384, Sigma, USA) 6mg (lmg/ml), 600-700 μ 1 rat serum (10-12%) to form a DMEM culture solution. This solution is called the First-Day culture solution or the Overnight culture solution. [0033] Isolated islets (1000) are put into this culture solution at a 37 C, 5% CO ₂ humidity incubator for overnight culture. On the second day, it is spread out on a petri dish with 150 μ 1 of matrigel (Collaborative Biomedical Products, USA) where it is consolidated for 30 minutes at 37 C.

[0034] Overnight cultured islets are gathered in a 1.5ml tube (Eppendorf) and most of the supernatant is taken off. 150 μ 1 of matrigel is added and mixed well. The well mixed gel and islets are put and spread out onto a Petri dish of which the floor is well coated gel. It is consolidated for 30 minutes at 37 C. IOng of PDGF (platelet derived growth factor) (p-8147, Sigma, USA) and lOOng of thrombin (T-4393, Sigma, USA) is added into 4.5ml of the First-Day culture solution. This is called the Second-Day culture solution. The gel

and islets are consolidated for 30 minutes and put into this culture solution after which it is cultured and proliferated at 37 C, CO ₂ (5%) humidity incubator.

[0035] On the third day, 37.5ng of HGF (Lot. 902287, Sigma, USA) is added to

4.5ml of the Second-Day culture solution to the third day solution. Third day solution is used for culture and proliferation.

[0036] The fourth day culture solution consists of 4.5ml of third day solution and

0.0151 μ of growth hormone (HG) (S8648, Sigma, USA) which is used for culture and proliferation on the fourth day.

[0037] Fifth day culture solution contains 4.5ml of fourth day culture solution and

150ng of progesterone (p-6149, Sigma, USA) and is used for culture and proliferation on the fifth day.

[0038] On the sixth day, the proliferated islets must be picked out from the matrigel and gathered. To do this, the culture solution is removed and 400 μ 1 of dispase (BD,

USA) and 300 μ 1 of DMEM is added before incubating for 10 minutes at 37 C, The islets and gel are separated by aspirating one or two times. The islets and gel are put into a 1.5ml tube (Eppendorf) and 500 μ 1 of 10% serum DMEM is added and mixed well. It is then put into a centrifugal separator for 5~10 seconds at lOOOrpm after which the supernatant is discarded.

[0039] EXAMPLE 2 - Synthesis of novel proteins in proliferated islets

[0040] 2-D gel electrophoresis procedure

[0041] 1) Place islets in a tube and centrifuge at lOOOrpm for 5 minutes.

[0042] 2) Discard supernatant, add PBS and centrifuge for 5 minutes at lOOOrpm.

Discard supernatant once again.

[0043] 3) Like this, wash islets with PBS 3 times.

[0044] 4) Add three times the sample volume of lysis buffer and sonicate on ice with tap. (Lysis buffer: 4OmM Tris, 0.1% Triton X-100 -> pH 8.0, discard cell membrane with detergent)

[0045] 5) Centrifuge for 5 minutes at 5000rpm. Mix 100 μ 1 of supernatant with three times the volume (300 μ 1) of TCA/acetone and leave for at least three hours or overnight. (2Og TCAC Trichloroacetic acid / 150ml acetone)

[0046] 6) Centrifuge for 30 minutes at 1200rpm and discard supernatant. Add acetone and centrifuge for 30 minutes at 1200rpm and discard supernatant again.

[0047] 7) Dry thoroughly and gather only the pure protein.

[0048] 8) Dissolve in rehydration solution (6M Urea, 2M thiourea, 2% CHAPS) and measure the amount of protein.

[0049] 9) Set the same protein amount and separate proteins according to protein charge through iso-electric focusing.

[0050] 10) Separate protein with SDS-PAGE according to the protein molecular weight.

[0051] 1 1 ) Detect protein by silver staining.

[0052] 12) Analyze the protein spots with image master program.

[0053] 13) Digest protein to make peptide. Observe using MALDI-TOF and predict protein through database searching.

[0054] 14) Partial amino acid sequence and compare results with results from

MALDI-TOF and identify protein.

[0055] Differences in protein expression were observed between the PI and freshly isolated islets (FI). Proteins from PI and freshly isolated islets (FI) were extracted, and separated using 2-dimensional electrophoresis. The results are shown in Figures 1 and 2.

The proteins only produced by FI are circled. During proliferation these proteins are diminished from the cytoplasm. The proteins only produced in PI are circled.

[0056] B-actin, which can only be seen in FI and disappears during proliferation, exists in a free state in the cytoplasm. During proliferation, B-actin is polymerized into filaments. Ck-8 is also another protein that disappears during proliferation. Albumin facilitates the influx of fatty acids into the cytoplasm by producing co-receptors in the membrane. In summary, proteins that exist in FI but do not in PI are not produced anymore or are produced very slowly during proliferation. On the other hand, of the proteins that are synthesized during proliferation, some have been reported to play roles in cell growth, and some are as yet uncharacterized.

[0057] EXAMPLE 3 - Function of PI to recover and maintain islet structure, function and viability after transplantation.

[0058] Advantageous expression of stress resistance genes in PI cells helps the islet to maintain its islet structure and viability after transplantation with the PIs. Islets in the pancreas have longevity and the ability to stay functional because of the many blood vessels that penetrate the islet, supplying abundant amounts of oxygen and nutrients. However, islet cells, especially β-cells, express only a small amount of anti-apoptotic genes, such as Bcl-2, making them easily susceptible to damage by cytotoxic radicals. Consequently, the transplantation of these islets into an organ other than the pancreas results in rapid decrease of function and subsequent destruction. Alternatively, the transplantation of PIs results in high survival rate.

[0059] One reason for the advantageous results obtained with using the PI cells is related to the concept that viability of cells, including islets, depends largely on the degree of expression of genes that encode proteins that protect the cells from a variety of stress, such as oxidative stress. Well-known genes include without limitation the anti- apoptotic gene Bcl-2 and antioxidant genes such as Mn-SOD, glutathione peroxidase GPX-I, A20 catalase and heme oxygenase (HO-I).

[0060] Expression of MNSOD, CuZnSOD and HO-I is increased in the presence of oxygen. Therefore, the expression of both SOD and HO-I during β-cell growth in hyperglycemic pancreatectomized animals and Zucker rat are different from those islets grown in vitro.

[0061] Hydrogen peroxide is a weak oxidant compared with the superoxide and hydroxyl radical. However, toxic hydroxy! radicals are formed from hydrogen peroxide. See chart below.

[0062] Consequently, in order to minimize damage to aconitase by superoxide, the superoxide must be quickly transformed into the weaker radical, H ₂ O ₂ , by the enzyme

Mn-SOD. Fortunately, MnSOD is expressed richly in PIs, similar to that of normal islets, enabling the rapid transformation of superoxide into H ₂ O ₂ . This is not the end, as H ₂ O ₂ can be easily transformed into hydroxyl radical, another powerful radical in the absence of GPX-I . GPX-I is needed in order to transform H ₂ O ₂ into H ₂ O. Pi's also synthesize

GPX-I in large quantities, enabling the speedy transformation of H ₂ O ₂ into H ₂ O and preventing any loss of islet viability.

[0063] EXAMPLE 4 -The expression levels of these genes in both PIs and FIs were determined by 2-D electrophoresis.

[0064] Protocol for total RNA extraction

[0065] 1) Prepare l -lθxlθ ⁵ cell in 1.5ml tube. Centrifuge it to remove culture media

(13,000rpm, l Osec), and add ImI of easy-BLUE.

[0066] 2) Vigorously vortex in room temperature for lOsec.

[0067] 3) Add 200 μ 1 of Chloroform and apply vortex

[0068] 4) After centrifuging the solution at 13,000rpm for 10 min, transfer 400μl of the upper fluid to an empty 1.5ml tube.

[0069] 5) Add 400μl of isopropanol (2-propanol) and mix it well by inverting the tube 2-3 times. Leave it for 10 min at room temperature.

[0070] 6) After centrifuging the solution at 13,000rpm (4°C) for 5 min, remove the upper layer to obtain RNA pellet.

[0071] 7) Add I mI of 75% EtOH and mix solution well by inverting the tube 2-3 times. Centrifuge the mixtures for 5 min at 10,000rpm (4°C). Discard the upper layer and dry the remaining RNA pellet.

[0072] 8) Dissolve RNA using 20-50μl of DEPC treated distilled water.

[0073] The results are shown in Figure 3.

[0074] Mn-SOD, A20, Bcl-2 expression levels are similar in both PI and FI with high levels of Mn-SOD and A20 expression. HO-I was expressed higher in FI than PI, but without significance, and GPX-I was expressed higher in PI than FI with significance.

HO-I is sensitive to and is expressed proportional to oxygen levels. However, GPX-I expression is less sensitive towards oxygen than HO-I and plays a more crucial role in maintaining cell viability. Thus, without the presence of GPX-I, cellular H ₂ O ₂ transforms into the powerful radical, hydroxyl radical, resulting in a significant loss of cell viability and eventual cell death. In PIs, GPX-I is expressed 2-fold compared to FIs, preventing β- cell death and maintaining the function of islets even when transplanted in a low-oxygen environment. Thus, PIs are advantageously useful for transplantation.

[0075] Figures 4A and 4B show that the transplantation of FI results in scarce distribution of insulin-secreting β-cells, causing a significant reduction in insulin secretion (A). In contrast, the transplanted PIs have a highly concentrated distribution of β-cells and compact structure, indicating a much higher insulin-secreting capacity (B).

[0076] Conclusion

[0077] 1) PIs have a blood glucose controlling ability, indicating the possibility of partially alleviating the problem of insufficient islet supply.

[0078] 2) The concept of a living donor may be realized and only a single transplantation of a full islet mass may be required in order to recover and maintain normoglycemia, greatly reducing the emotional burden and risk for the operating medical doctor.

[0079] 3) PIs are able to reserve their structure and function even though they are transplanted in an ectosite. This means that the curing effects of the islet transplantation could last longer than FIs.

[0080] 4) PIs stimulate the regeneration and neogenesis of pancreatic β-cells, prolonging the curing effect of islet transplantation indefinitely. Immunosuppressants may be used only for a short period, thus pacifying economic burden.

[0081] 5) Based on the data, PIs must be used for islet transplantation instead of FIs in the future.

[0082] EXAMPLE 5 -Islet regeneration/neogenesis

[0083] The phenomenon of islet regeneration and neogenesis in the pancreas after the recovery and maintenance of normoglycemia by proliferated islet transplantation (PIT) was confirmed. The number of islets in the pancreas was counted and examined to determine whether there was an increase. In addition, the transplanted islets were removed after a certain period of normoglycemia was sustained, and followed up by measuring the blood glucose levels to make certain that the transplantation of PIs stimulates pancreatic islet regeneration/neogenesis.

[0084] Results

[0085] 1) Pancreatic islet EQ number of mice transplanted with PI or FI. In mice transplanted with FI, the number of pancreatic islets did not increase as the period of normoglycemia prolonged. In contrast, mice transplanted with PI showed increasing islet

EQ number in proportion with the duration of normoglycemia achieved (Figure 6).

[0086] 2) Blood glucose level changes after removal of PI or FI (Figures 7 and 8).

[0087] 3) Plasma glucose profile after removal of PIs: Blood glucose levels increased very slowly and the degree of increase was lower as the duration of normoglycemia achieved was prolonged. Finally, as seen in Figures 7 and 8, blood glucose level is normoglycemia as the duration was over 5~7 months even though the transplanted PI were removed.

[0088] Islet neogenesis could not be seen in mice that achieved normoglycemia for only 2-5 months after PIT (Figures 9A-9C).

[0089] EXAMPLE 6 - In vitro proliferated islets exhibit diverse and active metabolism including creating and secreting energy and metabolites which enable them to absorb

nutrients from the culture solution. This change is likely caused by big difference in surface structure of the proliferated islets and that of fresh islets. For this, observation has been made of islets cultured overnight and islets proliferated for five days. SAM and

TEM were used.

[0090] Results:

[0091] Fresh islets display smoother surface than proliferated islets. The surface of proliferated islets is very irregular with many small-pocket-like-looking structures. These pocket structures are connected with the cell membrane (Figures 10A-10B and UA-

HB).

[0092] One of the proteins generated only in proliferated islets is SmCX. This protein is known as a DNA binding protein but its specific function is unknown.

[0093] In one aspect, a method of making the proliferated islet cells is described in

U.S. Patent No. 6,506,599, the contents of which are incorporated by reference herein in its entirety.

[0094] AU of the references cited herein are incorporated by reference in their entirety.

* * * * *

[0095] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.