WO2018216021A1 - Method of promoting in vitro beta cell proliferation - Google Patents

Abstract

A method of promoting in vitro beta cell proliferation is provided. The method comprises subjecting beta cells to an effective amount of at least one growth factor expressed by pancreatic pericytes during the neonatal period, thereby promoting in vitro beta cell proliferation.

Description

METHOD OF PROMOTING IN VITRO BETA CELL PROLIFERATION

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of expanding pancreatic beta cells and to uses thereof for treating diabetes.

Diabetes is a chronic disease that is now reaching pandemic proportions. It occurs from a deficiency or functional impairment of insulin-producing pancreatic β-cells, alone or in combination with insulin resistance. Type 1 diabetes (T1D) is characterized by decreased β-cell mass due to immuno-destruction of these cells. However, even when this response is inhibited, β-cell mass can remain low and leave T1D patients dependent on external insulin administration.

Type 2 diabetes (T2D) manifests itself in individuals who lose the ability to produce sufficient amounts of insulin to maintain normoglycaemia in the face of insulin resistance. As pancreatic β-cell mass is reduced in both T1D and T2D mellitus, β-cell replacement represents an attractive approach for treating diabetes. However, such therapy is currently limited by shortage in available β-cells. Developing approaches for expanding these cells has proven challenging as β-cells normally undergo replication only in the embryonic and neonatal periods. Thus, elucidating factors controlling neonatal β-cells proliferation is instrumental for developing protocols for expanding β-cells for medical use. Pancreatic pericytes are a central component of β-cell microenvironment.

Sasson et al [Diabetes 2016 Oct; 65(10): 3008-3014] teach that pericytes are required for β-cell replication during embryonic development.

Epshtein et al [Molecular Metabolism 2017 Oct;6(10):1330-1338] teach that pericytes are required for β-cell replication during the neonatal period.

Hughes, A. et al., 2014. Journal of Endocrinology, 221(2), pp.R41-8 teaches that IGF-1 is a β-cell growth and an anti-apoptotic factor.

Additional background art includes Silva Krause et al., Journal of Endocrinology (2012) 214, 301-311.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided an in vitro method of promoting proliferation of insulin-producing cells, the method comprising subjecting insulin-producing cells to an effective amount of at least one growth factor expressed by pancreatic pericytes at the neonatal period under conditions which allow the proliferation of the insulin secreting cells by at least two fold, with the proviso that the at least one growth factor is not insulin-like growth factor 2 (IGF2), thereby promoting proliferation of insulin-producing cells.

According to another aspect of some embodiments of the present invention there is provided an in vitro method of promoting proliferation of insulin-producing cells, the method comprising subjecting insulin-producing cells to an effective amount of at least two growth factors expressed by pancreatic pericytes at the neonatal period, thereby promoting proliferation of insulin-producing cells.

According to another aspect of some embodiments of the present invention there is provided a method of promoting proliferation of insulin-producing cells, the method comprising culturing the insulin-producing cells in a conditioned medium of neonatal pericytes, thereby promoting proliferation of insulin-producing cells.

According to another aspect of some embodiments of the present invention there is provided in vitro expanded cells obtainable according to the methods described herein.

According to another aspect of some embodiments of the present invention there is provided a use of the described herein in the manufacture of a medicament for treating insulin deficiency.

According to another aspect of some embodiments of the present invention there is provided a method of generating a conditioned medium useful for proliferation of insulin- producing cells the method comprising:

(a) culturing neonatal pericytes in a medium for a predetermined time so as to generate a medium comprising growth factors secreted from the neonatal pericytes; and

(b) isolating the medium comprising growth factors from the neonatal pericytes, thereby generating the conditioned medium.

According to some embodiments of the invention, the at least one growth factor is selected from the group consisting of Midkine (MDK), Interleukin 6 (IL-6), Pleiotrophin (Ptn) and human CXCL2.

According to some embodiments of the invention, the at least two growth factors are selected from the group consisting of IGF2, Midkine (MDK), Interleukin 6 (IL-6), Pleiotrophin (Ptn) and human CXCL2.

According to some embodiments of the invention, the growth factor is a recombinant growth factor.

According to some embodiments of the invention, the subjecting is effected by culturing the cells in the presence of the growth factor or a functional equivalent thereof. According to some embodiments of the invention, the subjecting is effected by culturing the cells in a conditioned medium comprising the growth factor.

According to some embodiments of the invention, the conditioned medium is generated by culturing neonatal pericytes.

According to some embodiments of the invention, the culturing is effected for at least 10 passages.

According to some embodiments of the invention, the subjecting is effected by co- culturing the cells with an additional population of cells that secrete the growth factor.

According to some embodiments of the invention, the additional population of cells comprises neonatal pericytes.

According to some embodiments of the invention, the subjecting is effected by expressing in the cells the growth factor or a functional equivalent thereof.

According to some embodiments of the invention, the at least one growth factor comprises at least 2 and no more than 5 growth factors.

According to some embodiments of the invention, the at least one growth factor comprises MDK and IL-6; MDK and Ptn; MDK and human CXCL2; Interleukin 6 (IL-6) and Pleiotrophin (Ptn); or Interleukin 6 (IL-6) and human CXCL2.

According to some embodiments of the invention, the at least two growth factors comprise IGF2 and Midkine (MDK); IGF2 and Interleukin 6; IGF2 and Pleiotrophin (Ptn); IGF2 and human CXCL2; MDK and IL-6; MDK and Ptn; MDK and human CXCL2; Interleukin 6 (IL-6) and Pleiotrophin (Ptn); Interleukin 6 (IL-6) and human CXCL2 or IGF2 and human CXCL2.

According to some embodiments of the invention, the insulin-producing cells are pancreatic beta cells.

According to some embodiments of the invention, the pancreatic beta cells are cadaveric cells.

According to some embodiments of the invention, the insulin-producing cells are stem cell-derived.

According to some embodiments of the invention, the insulin-producing cells are adult cells.

According to some embodiments of the invention, the insulin-producing cells are fetal cells.

According to some embodiments of the invention, the insulin-producing cells are human cells. According to some embodiments of the invention, the insulin deficiency is associated with TlDM or T2DM.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-C. Culturing neonatal pancreatic pericytes:

A) Flow-cytometry analysis of digested pancreatic tissue. Left, Dotplot showing the presence of a yellow fluorescent cell population (gated green cells; 'Nkx3.2/YFP⁺ cells') in the pancreas of x?.2-Cre; ?2<5-YFP at postnatal day 5 (p5). Right, Green histogram ('+Primary antibody') showing the staining of Nkx3.2/YFP⁺ cells (as gated in left panel) for the pericytic marker PDGFRp. Gray histogram showing the analysis of Nkx3.2/YFP⁺ cells without the addition of the primary antibody ('Staining control'). The number represents the percentage of PDGFRP-stained cells from the total Nkx3.2/YFP⁺ cell population (as indicated by a horizontal line). Note that the vast majority of Nkx3.2/YFP⁺ cells express this pericytes' marker.

B) Schematic illustration of cultured neonatal pancreatic pericytes. Pancreatic tissues of Nkx3.2-QcQ,R26-Y¥V p5 pups were dissected and digested to obtain a single cell suspension. Pericytes were FACS sorted based on their yellow fluorescence (as shown in A', Nkx3.2/YFP⁺ cells), and cultured in complete DMEM medium. During the cells' fourth passage, their conditioned media were collected.

C) Cultured neonatal pancreatic pericytes. A yellow fluorescent image (showed in green for easier visualization) overlaid on top of a brightfield image of cultured Nkx3.2/YFP⁺ cells (as described in Β') during their fourth passage. Representative field is shown. Note extension of cytoplasmic process by cultured cells.

FIGs. 2A-E Increased β-cell proliferation upon exposure to pericyte-conditioned medium

A) Tetracycline-treated pTC-tet cells were cultured in either control (complete DMEM; 'Control medium') or neonatal pericyte-conditioned ('Conditioned medium'; described in Figure

IB) medium, both supplemented with tetracycline. After incubation for 96 hours, cells were fixed and stained for the proliferative marker Ki67. Left, representative dotplots showing flow- cytometry analysis of Ki67 expression by pTC-tet cells. Gated are Ki67⁺ cells; the numbers represent the percentage of gated cells out of the analyzed cell population. Right, Bar diagrams (mean + SD) represent the percentage of Ki67⁺ cells. N = 3. ***P < 0.005 (Student's t-test), as compared to the control medium. A representative of three independent experiments is shown.

B) Isolated islets from 3 -month-old wild-type mice were cultured in either control (complete DMEM; 'Control medium') or neonatal pericyte-conditioned ('Conditioned medium'; described in Fig. IB) medium for 24 hours. Islets were dispersed to single cells, fixed, and stained for insulin and the proliferative marker Ki67. Left, representative dotplots showing flow- cytometry analysis of Ki67 expression by insulin^"1" cells. Gated are Ki67⁺ cells; the numbers represent the percentage of gated cells out of the total insulin^"1" cell population. Right, Bar diagrams (mean + SD) represent the percentage of Ki67⁺ out of the total number of insulin^"1" cells. N = 4. ***P < 0.005 (Student's t-test), as compared to the control media. A representative of four independent experiments is shown.

C) Isolated islets were cultured as described in B' for 72 hours. Islets were dispersed to single cells, fixed and stained for insulin. Bar diagrams (mean + SD) represent the relative number of insulin^"1" cells, normalized to islets incubated with control medium. N = 3-4. *P < 0.05 (Student's t-test).

D) Control (complete DMEM) and neonatal pericyte-conditioned ('conditioned medium' ; as described in Figure IB) medium were heated to 62 °C for 20 minutes. Isolated islets from 3 -month-old wild-type mice were cultured in heated media for 24 hours. Islets were dispersed to single cells, fixed and stained for insulin and the proliferative marker Ki67. Bar diagrams (mean + SD) represent the percentage of Ki67⁺ out of the total number of insulin^"1" cells (gated as shown in B'). N = 3 - 4. NS = non-significant (Student's t-test).

E) Tetracycline-treated pTC-tet cells were incubated with control (complete DMEM; tetracycline- supplemented) medium or neonatal pericyte-conditioned medium ('Conditioned medium', tetracycline- supplemented) for 96 hours. The conditioned medium was supplemented with either anti- βΐ integrin blocking antibody ('Anti- βΐ integrin') or control IgM. Bar diagrams (mean + SD) represent the relative cell number, normalized to cells incubated with control medium. N = 3. *P < 0.05, ***P < 0.005, NS = non- significant (Student's t-test).

FIGs. 3A-F. Partial depletion of pancreatic pericytes in DT-treated x?.2-Cre;iDTR pups. x?.2-Cre;iDTR transgenic pups and littermate controls (carrying the iDTR transgene, but not the x?.2-Cre transgene; 'Control [iDTR]') were i.p. injected with 0.25 ng/gr body weight DT at p3 and analyzed at p5 ('DT p3→p5') or p21 ('DT p3→p21').

A) Schematic illustration of mouse treatment.

B) Bar diagram (mean + SD) showing the relative body weight of DT-treated Nkx3.2- Cre;iDTR (empty bars) and control (black bars, set to T) littermates at p5 and p21. n = 5.

C) Pancreatic tissues of DT-treated p5 x?.2-Cre;iDTR (right) and control (left) mice were stained for NG2 (red) to label pericytes, PECAM1 (green) to label endothelial cells, and insulin to label β-cells. White lines demarcate the outer border of the insulin^"1" area. Note that all capillaries in control islets contained both endothelial cells and pericytes, whereas some capillaries in x?.2-Cre;iDTR islets contained only endothelial cells. Representative fields are shown. The same imaging parameters were used to analyze Mx?.2-Cre;iDTR and control tissues.

D, E) Bar diagrams (mean + SD) showing decreased intra-islet pericyte density (D), but not endothelial density (E), in DT-treated p5 Mx?.2-Cre;iDTR mice (empty bars) compared with a control (black bars, set to T). Pancreatic tissues were stained as described in C, and the relative ratio of NG2⁺ or PECAM1^"1", and the Insulin^"1" area for each islet was calculated. At least 30 islets per mouse, from sections at least 50 μιη apart, were analyzed. N = 3. p < 0.005 (Student's t-test), as compared to control littermates.

F) Pancreatic tissues of DT-treated p5 Mx?.2-Cre;iDTR (right) and control (left) mice were stained for aSMA (red) to label vSMCs, and PECAM1 (green) to label endothelial cells. Representative fields are shown. The same imaging parameters were used to analyze Nkx3.2- Cre;iDTR and control tissues.

FIGs. 4A-B. Reduced neonatal β-cell proliferation rates upon pericyte depletion. Nkx3.2- Cre;iDTR transgenic pups and littermate controls (carrying the iDTR transgene, but not the Mx?.2-Cre transgene; 'Control [iDTR]') were i.p. injected with 0.25 ng/gr body weight DT at p3 and analyzed at p5 ('DT p3→p5').

A) Pancreatic tissues were stained for insulin to label β-cells (red) and Ki67 (green) to mark proliferative cells. Left, representative fields showing immunofluorescence analysis of Ki67 and insulin. Right, Bar diagrams (mean + SD) represent the percentage of Ki67⁺ cells out of the total number of insulin^"1" cells in DT-treated p5 Mx?.2-Cre;iDTR mice (empty bars) compared with control (black bars, set to T), stained as shown in left panels. At least 300 insulin^"1" cells were analyzed for each mouse. The same imaging parameters were used to analyze x?.2-Cre;iDTR and control tissues. N = 3. ***P < 0.005 (Student's t-test).

B) Pancreatic tissues of DT-treated p5 x?.2-Cre;iDTR (middle panel) and control (left panel) were subjected to TUNEL assay (green) to identify dying cells, and were stained for insulin (red) to identify β-cells. The right panel shows similarly stained non-transgenic pancreatic tissue pre-treated with DNase to induce DNA breaks, which served as a positive control for the TUNEL assays ('staining control'). Representative fields are shown. The same imaging parameters were used to analyze Mx?.2-Cre;iDTR and control tissues.

FIG. 5 are graphs illustrating temporal expression of growth factors by the neonatal pancreatic pericytes. Upper left panel, Age-dependent β-cell replication in neonatal mice (adapted from Gregg, B. E., et al. 2012, J. Clin. Endocrinol. Metab. 97, 3197-3206 and Chamberlain, C. E.,et al., (2014) J. Clin. Invest. 124, 4093-4101). Note a peak at postnatal day 5. Remaining panels, Expression levels of indicated growth factors at pancreatic pericytes isolated from pO, p5, pl4 and p21 mouse pups.

FIG. 6 is a graph illustrating increased β-cell proliferation upon exposure to selected pericytic growth factors. Tetracycline-treated pTC-tet cells were cultured in either control (non- supplemented DMEM) medium or media supplemented with indicated growth factors. After incubation for 96 hours, cells were fixed and stained for the proliferative marker Ki67. Bar diagrams (mean SD) represent the percentage of Ki67⁺ cells. N = 4. **P<0.01, ***P<0.005 (Student's i-test), as compared to the control medium. NS = not significant.

EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of expanding pancreatic beta cells and to uses thereof for treating diabetes.

The principles and operation of the expanded and re-differentiated isolated population of adult beta cells according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Type I diabetes is caused by the autoimmune destruction of the pancreatic islet insulin- producing beta cells. Insulin administration does not prevent the long-term complications of the disease, since the optimal insulin dosage is difficult to adjust. Replacement of the damaged cells with regulated insulin-producing cells is considered the ultimate cure for type 1 diabetes. Pancreas transplantation has been successful but is severely limited by the shortage of donors. With the development of new islet isolation and immunosuppression procedures, significant success has been reported using islets from 2-3 donors per recipient (Shapiro AM, Lakey JR, Ryan EA et al. New Engl J Med 2000;343:230-238). This progress underscores the urgent need for developing alternatives to human pancreas donors, namely abundant sources of cultured human pancreatic β cells for transplantation.

While reducing the present invention to practice, the present inventors have uncovered a novel approach for ex-vivo expansion of isolated adult islet beta cells.

Specifically, the present inventors have found that neonatal pancreatic pericytes secretes factors that stimulate β-cell proliferation. Five growth factors that are expressed by neonatal pancreatic pericytes, specifically during periods of extensive β-cell proliferation have now been identified.

Whilst reducing the present invention to practice, the present inventors demonstrated that two of the identified growth factors (namely insulin-like growth factor 2 (IGF-2) and Pleiotrophin) enhanced proliferation of β cell line (Figure 6).

Accordingly, the use of these growth factors for in vitro expansion of human β-cells derived from either cadaveric islets or stem cells differentiation protocols, prior to their use for cell replacement therapy, is now contemplated as a potential cure to diabetes.

Thus, according to one aspect of the present invention there is provided an in vitro method of promoting proliferation of insulin-producing cells, the method comprising subjecting insulin-secreting cells to an effective amount of at least one growth factor expressed by pancreatic pericytes at the neonatal period under conditions which allow the proliferation of the insulin secreting cells by at least two fold, with the proviso that the at least one growth factor is not insulin growth factor 2 (IGF2), thereby promoting proliferation of insulin-producing cells.

As used herein, the phrase "insulin-producing cells" refers to cells expressing insulin polypeptides or peptides derived therefrom. In one embodiment, the insulin-producing cells are capable of secreting insulin in response to elevated glucose concentrations and express at least one, at least two, at least three, at least four typical of pancreatic beta cell markers. Examples of beta cell markers include, but are not limited to, insulin, pdx, Ηηί3β, PCl/3, Beta2, Nkx2.2, GLUT2 and PC2. According to one embodiment, the insulin-producing cells are derived from the pancreas- i.e. pancreatic beta cells.

Pancreatic beta cells may be obtained from any autologous or non-autologous (i.e., allogeneic or xenogeneic) mammalian donor (e.g. human, mouse, monkey, rat). For example, pancreatic beta cells may be isolated from a human cadaver.

In one embodiment, the pancreatic beta cells comprise post-natal (e.g., non-embryonic) pancreatic endocrine cells. Thus, for example adult pancreatic beta cells are contemplated.

In another embodiment, the pancreatic beta cells are fetal pancreatic beta cells.

The pancreatic beta cells may be a homogeneous population of pancreatic beta cells or may be comprised in a cell population of other cells of pancreatic islets including for example alpha cells that produce glucagon; and/or delta cells (or D cells) that produce somatostatin; and/or PP cells that produce pancreatic polypeptide.

Methods of isolating islets are well known in the art. For example, islets may be isolated from pancreatic tissue using collagenase and ficoll gradients.

Preferably the pancreatic beta cells of the present invention are dispersed into a single cell suspension - e.g. by the addition of trypsin or by trituration.

The pancreatic beta cells may be further isolated being substantially free from other substances (e.g., other cells, proteins, nucleic acids, etc.) that are present in its in-vivo environment e.g. by FACs sorting.

It will be appreciated that pancreatic beta cells may also be generated ex-vivo - for example by differentiating stem cells (e.g. human stem cells) or progenitor cells (e.g. pancreatic progenitor cells).

As used herein, the phrase "stem cells" refers to cells which are capable of remaining in an undifferentiated state (i.e. "pluripotent stem cells") for extended periods of time in culture until induced to differentiate into other cell types having a particular, specialized function (i.e., "fully differentiated" cells).

The stem cells of the present invention can be adult tissue stem cells. As used herein, "adult tissue stem cells" refers to any stem cell derived from the postnatal animal (especially the human). The adult stem cell is generally thought to be a multipotent stem cell, capable of differentiation into multiple cell types. Adult stem cells can be derived from an adult tissue such as adipose tissue, skin, kidney, liver, prostate, pancreas, intestine, and bone marrow.

Methods of isolating adult tissue stem cells are known in the arts and include, for example, those disclosed by Alison, M.R. [Tissue-based stem cells: ABC transporter proteins take center stage. J Pathol. 2003 200(5): 547-50], Cai, J. et al., [Identifying and tracking neural stem cells. Blood Cells Mol Dis. 2003 31(1): 18-27] and Collins, A.T. et al., [Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(l)-integrin expression. J Cell Sci. 2001; 114(Pt 21): 3865-72].

Generally, isolation of adult tissue stem cells is based on the discrete location (or niche) of each cell type included in the adult tissue, i.e., the stem cells, the transit amplifying cells and the terminally differentiated cells [Potten, C. S. and Morris, R. J. (1988). Epithelial stem cells in vivo. J. Cell Sci. Suppl. 10, 45-62]. Thus, an adult tissue such as, for example, prostate tissue is digested with CoUagenase and subjected to repeated unit gravity centrifugation to separate the epithelial structures of the prostate (e.g., organoids, acini and ducts) from the stromal cells. Organoids are then disaggregated into single cell suspensions by incubation with Trypsin/EDTA (Life Technologies, Paisley, UK) and the basal, CD44-positive, stem cells are isolated from the luminal, CD57-positive, terminally differentiated secretory cells, using anti-human CD44 antibody (clone G44-26; Pharmingen, Becton Dickinson, Oxford, UK) labeling and incubation with MACS (Miltenyi Biotec Ltd, Surrey, UK) goat anti-mouse IgG microbeads. The cell suspension is then applied to a MACS column and the basal cells are eluted and re-suspended in WAJC 404 complete medium [Robinson, E.J. et al. (1998). Basal cells are progenitors of luminal cells in primary cultures of differentiating human prostatic epithelium Prostate 37, 149-160].

In one embodiment, the stem cells utilized by the present invention are BM-derived stem cells including hematopoietic, stromal or mesenchymal stem cells (Dominici, M et al., 2001. Bone marrow mesenchymal cells: biological properties and clinical applications. J. Biol. Regul. Homeost. Agents. 15: 28-37). BM-derived stem cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullar spaces.

In another embodiment, the insulin-producing cells are generated from embryonic stem cells or induced pluripotent stem cells.

The phrase "embryonic stem cells" refers to embryonic cells which are capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm), or remaining in an undifferentiated state. The phrase "embryonic stem cells" may comprise cells which are obtained from the embryonic tissue formed after gestation (e.g., blastocyst) before implantation of the embryo (i.e., a pre-implantation blastocyst), extended blastocyst cells (EBCs) which are obtained from a post-implantation/pre-gastrulation stage blastocyst (see WO2006/040763) and embryonic germ (EG) cells which are obtained from the genital tissue of a fetus any time during gestation, preferably before 10 weeks of gestation.

According to a particular embodiment, the embryonic stem cells are generated without destruction of a human embryo. Induced pluripotent stem cells (iPS; embryonic-like stem cells), are cells obtained by de- differentiation of adult somatic cells which are endowed with pluripotency (i.e., being capable of differentiating into the three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm). According to some embodiments of the invention, such cells are obtained from a differentiated tissue (e.g., a somatic tissue such as skin) and undergo de-differentiation by genetic manipulation which re-program the cell to acquire embryonic stem cells characteristics. According to some embodiments of the invention, the induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in a somatic stem cell.

Various protocols are known in the art for differentiating stem cells to become insulin- producing cells. The protocols may be such that the differentiated cells store insulin and secrete insulin in response to glucose. Methods of increasing beta cell insulin content may include increasing insulin transcription and/or post transcriptional control and/or increasing translation and/or post-translational control. Methods of increasing beta cell insulin content may also include enhancing insulin storage and/or retarding insulin breakdown. Methods of increasing sensitivity to glucose may include increasing the expression of glucose transporters.

According to one embodiment the stem cells are differentiated using a beta cell differentiation promoting agent.

As used herein a "beta cell differentiation promoting agent" refers to a molecule (e.g., a proteinaceous or nucleic molecule) which is able either alone or in combination with other beta cell differentiation promoting agents to further differentiate stem or progenitor cells.

Examples of beta cell differentiation promoting agents include but are not limited to Activin A, Atrial Natriuretic Peptide, Betacellulin, Bone Morphogenic Protein (BMP-2), Bone Morphogenic Protein (BMP-4), C natriuretic peptide (CNP), Caerulein, Calcitonin Gene Related Peptide (CGRP-ax), Cholecystokinin (CCK8-amide), Cholecystokinin octapeptide (CCK8- sulfated), Cholera Toxin B Subunit, Corticosterone (Reichstein's substance H), Dexamethasone, DIF-1, Differanisole A, Dimethylsulfoxide (DMSO), EGF, Endothelin 1, Exendin 4, FGF acidic, FGF2, FGF7, FGFb, Gastrin I, Gastrin Releasing Peptide (GRP), Glucagon-Like Peptide 1 (GLP-1), Glucose, Growth Hormone, Hepatocyte Growth Factor (HGF), IGF-1, IGF-2, Insulin, KGF, Lactogen, Laminin, Leu-Enkephalin, Leukemia Inhibitory Factor (LIF), Met-Enkephalin, n Butyric Acid, Nerve Growth Factor (.beta.-NGF), Nicotinamide, n-n-dimethylformamide (DMF), Parathyroid Hormone Related Peptide (Pth II RP), PDGF AA+PDGF BB MIX, PIGF (Placental GF), Progesterone, Prolactin, Putrescine Dihydrochloride Gamma- Irradiated Cell Culture, REG1, Retinoic Acid, Selenium, Selenious Acid, Sonic Hedgehog, Soybean Trypsin Inhibitor, Substance P, Superoxide Dismutase (SOD), TGF-alpha, TGF-beta. sRII, TGF-beta.l, transferrin, Triiodothyronine (T3), Trolox, Vasoactive Intestinal Peptide (VIP), VEGF, Vitamin A and Vitamin E.

A beta cell differentiation promoting agent may also be a transcription factor. The term "beta cell differentiation transcription factor" as used herein is defined as any molecule, either a polypeptide or a nucleic acid expressing the polypeptide, which is involved in beta cell differentiation by functioning as a transcription factor. The transcription factor may also participate in additional mechanisms directed to development, metabolism or the like. Examples of beta cell differentiation transcription factor include, but are not limited to, NeuroD (GenBank Accession No. AAA93480), Pax6 (GenBank Accession No. AAK9584, Pax4 (GenBank Accession No. AAD02289, Nkx2.2 (GenBank Accession No. AAC83132), Nkx6.1 (GenBank Accession No. AAD11962, Is 1-1 (GenBank Accession No. NP002193, Pd-x (GenBank Accession No. AAA88820) or Ngn3 (GenBank Accession No. AAK15022) and homologues or orthologues of same.

As mentioned, in order for the insulin-producing cells of the present invention to proliferate they are subjected to an effective amount of at least one growth factor expressed by pancreatic pericytes at the neonatal period.

Preferably, the growth factor is one that is expressed at high levels by pancreatic pericytes, especially in ages of high β-cell replication rate (e.g. at least twice the amount, at least three times the amount or even at least four times the amount than the level of expression at ages of low β-cell replication rate).

An exemplary growth factor contemplated by the present invention includes a member of the Midkine family.

According to a particular embodiment, the Midkine family member is Midkine (also known as NEGF2, Uniprot No. P21741) having an amino acid sequence at least 90 %, 95, %, 96 %, 97 %, 98 %, 99 % 100 % identical/homologous over its length or functional region to a segment (preferably a continuous segment) of a wild-type Midkine (SEQ ID NO: 1). In another embodiment the Midkine family member is an orthologue of SEQ ID NO: 1.

According to another embodiment, the Midkine family member is pleiotriphin (also known as NEGFl; Uniprot No. P21246) having an amino acid sequence at least 90 %, 95, %, 96 %, 97 %, 98 %, 99 % 100 % identical/homologous over its length or functional region to a segment (preferably a continuous segment) of a wild-type pleiotrophin (e.g. SEQ ID NO: 2). In another embodiment the Midkine family member is an orthologue of SEQ ID NO: 2.

The Midkine family protein may be a functional variant of a Midkine family protein, including a functional variant of midkine, midkine-like protein, truncated midkine protein, or pleiotrophin. The functional variant may be modified by substitution, deletion, or addition of one or more amino acids relative to the non-modified protein. The functional variant of the Midkine family protein will exhibit a function of midkine (e.g enhancing cell proliferation, inhibiting apoptosis, binding to heparin, enhancing cell migration, or inducing cell differentiation). In one embodiment, the function of MK is enhancing cell proliferation).

Another exemplary growth factor contemplated by the present invention includes interleukin-6 (IL-6).

According to a particular embodiment, the IL-6 (also known as interferon B2, Uniprot No. P05231) has an amino acid sequence at least 90 %, 95, %, 96 %, 97 %, 98 %, 99 % 100 % identical/homologous over its length or functional region to a segment (preferably a continuous segment) of a wild-type P05231 (SEQ ID NO: 3). In another embodiment the IL-6 is an orthologue of SEQ ID NO: 1.

The IL-6 may be a functional variant of IL-6. The functional variant may be modified by substitution, deletion, or addition of one or more amino acids relative to the non-modified protein. The functional variant of IL-6 protein will exhibit a function of IL-6 (e.g enhancing cell proliferation).

Another exemplary growth factor contemplated by the present invention includes C-X- C Motif Chemokine Ligand 2 (CXCL2).

According to a particular embodiment, the CXCL2 (also known as GR02 oncogene, Macrophage Inflammatory Protein 2- Alpha, Melanoma Growth Stimulatory Activity Beta Uniprot No. P19875) has an amino acid sequence at least 90 %, 95, %, 96 %, 97 %, 98 %, 99 % 100 % identical/homologous over its length or functional region to a segment (preferably a continuous segment) of a wild-type P19875 (SEQ ID NO: 4). In humans, this protein is encoded by the CXCL2 gene. In another embodiment the CXCL2 is an orthologue of SEQ ID NO: 4.

The CXCL2 may be a functional variant of CXCL2. The functional variant may be modified by substitution, deletion, or addition of one or more amino acids relative to the non- modified protein. The functional variant of CXCL2protein will exhibit a function of CXCL2 (e.g enhancing cell proliferation).

Another exemplary growth factor contemplated by the present invention includes insulin-like growth factor 2 (IGF2).

According to a particular embodiment, the IGF-2 (Uniprot No. P01344) has an amino acid sequence at least 90 %, 95, %, 96 %, 97 %, 98 %, 99 % 100 % identical/homologous over its length or functional region to a segment (preferably a continuous segment) of a wild-type P01344 (SEQ ID NO: 5). In another embodiment the IGF-2 is an orthologue of SEQ ID NO: 5. The IGF-2 may be a functional variant of IGF-2. The functional variant may be modified by substitution, deletion, or addition of one or more amino acids relative to the non- modified protein. The functional variant of IGF-2 protein will exhibit a function of IGF-2 (e.g enhancing cell proliferation).

The growth factors of the present invention may be synthesized using any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of polypeptides from natural sources, or the chemical synthesis of polypeptides.

In cases where large amounts of the growth factors are desired, they can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al., (1990) Methods in Enzymol. 185:60-89, Brisson et al., (1984) Nature 310:511-514, Takamatsu et al., (1987) EMBO J. 6:307-311, Coruzzi et al., (1984) EMBO J. 3: 1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al., (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art. For example, the growth factors of the present invention are commercially available from BTC; R&D Systems, Minneapolis, MN.

The growth factors for promoting proliferation of insulin-producing cells may be provided to the insulin-producing cells per se (e.g. added to the culture medium as recombinant proteins). Alternatively, polynucleotides encoding same may be transfected into the insulin- producing cells. In this case, the polynucleotide agent is ligated in a nucleic acid construct under the control of a cis-acting regulatory element (e.g. promoter) capable of directing an expression of the growth factor in the insulin-producing cells in a constitutive or inducible manner.

The nucleic acid construct may be introduced into the insulin-producing cells of the present invention using an appropriate gene delivery vehicle/method (transfection, transduction, etc.) and an appropriate expression system. Examples of suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. Lipid-based systems may be used for the delivery of these constructs into the expanded adult islet beta cells of the present invention. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. Recently, it has been shown that Chitosan can be used to deliver nucleic acids to the intestine cells (Chen J. (2004) World J Gastroenterol 10(1): 112-116). Other non-lipid based vectors that can be used according to this aspect of the present invention include but are not limited to polylysine and dendrimers.

The expression construct may also be a virus. Examples of viral constructs include but are not limited to adenoviral vectors, retroviral vectors, vaccinia viral vectors, adeno-associated viral vectors, polyoma viral vectors, alphaviral vectors, rhabdo viral vectors, lenti viral vectors and herpes viral vectors.

A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-transcriptional modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. In addition, such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed. Preferably, the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the peptide variants of the present invention. Optionally, the construct may also include a signal that directs polyadenylation, as well as one or more restriction site and a translation termination sequence. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.

Preferably the viral dose for infection is at least 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,

10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵ or higher pfu or viral particles.

It will be appreciated that expression of more than one growth factor in the insulin- producing cells of the present invention may be desired, as further described herein below. Various construct schemes can be utilized to express more than one growth factor from a single nucleic acid construct.

For example, the two recombinant proteins can be co-transcribed as a polycistronic message from a single promoter sequence of the nucleic acid construct. To enable co- translation of two growth factors from a single polycistronic message, the first and second polynucleotide segments can be transcriptionally fused via a linker sequence including an internal ribosome entry site (IRES) sequence which enables the translation of the polynucleotide segment downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule including the coding sequences of both the first and the second growth factors will be translated from both the capped 5' end and the internal IRES sequence of the polycistronic RNA molecule to thereby produce both growth factors. Alternatively, the first and second polynucleotide segments can be translationally fused via a protease recognition site cleavable by a protease expressed by the cell to be transformed with the nucleic acid construct. In this case, a chimeric polypeptide translated will be cleaved by the cell expressed protease to thereby generate both growth factors.

Still alternatively, the nucleic acid construct of the present invention can include two promoter sequences each being for separately expressing both growth factors. These two promoters which may be identical or distinct can be constitutive, tissue specific or regulatable (e.g. inducible) promoters functional in one or more cell types.

As well as (or instead of) expression in the cells themselves, the growth factors, either alone or in combination, may be provided to the insulin-producing cells by addition to the incubating medium.

Preferably, the insulin-producing cells are cultured for at least two days, 5 days, 6 days, 7 days, 10 days, 14 days or more to allow for adequate cell proliferation.

An exemplary amount of Midkine (MDK) that can be added to the culture medium is 1- 100 μg/ml, more preferably between 1-30 μg/ml and more preferably between 3.0-8.0 μg/ml.

An exemplary amount of IL-6 that can be added to the culture medium is 0.05-8 ng/ml and more preferably between 0.2-0.8 ng/ml.

An exemplary amount of Pleiotrophin that can be added to the culture medium is between 1-50 μg/mL, and more preferably between 3-8 μg/ml.

An exemplary amount of human CXCL2 (hCXCL2) that can be added to the culture medium is between 1-100 ng/ml and more preferably between 3-15 ng/ml ranges.

An exemplary amount of IGF2 that can be added to the culture medium is between 0.2 - 100 ng/ml and more preferably between 2 - 10 ng/ml.

The insulin-producing cells of the present invention may be cultured for at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more passages whilst being subjected to the growth factors.

Examples of medium which can be used to propagate the insulin-producing cells include, but are not limited to DMEM, CMRL or RPMI.

Additional agents which can be included in the growth medium include glucose, serum, antibiotics and amino acids.

According to one embodiment the cell population is cultured for a time such that at least one population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least two population doubling occurs. In still another embodiment, the cell population is cultured for a time such that at least three population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least four population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least five population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least six population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least seven population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least eight population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least nine population doubling occurs. In another embodiment, the cell population is cultured for a time such that at least ten population doubling occurs.

In another embodiment, the growth factor (or combination thereof) is provided in a quantity that enhances proliferation of the insulin -producing cells.

An exemplary method for measuring the rate of cell proliferation is by staining for proliferative markers such as Ki67 and analyzing the cells by flow cytometry.

Optionally, the growth factor (or combination thereof) is provided in a quantity that is sufficient to increase insulin content in the adult islet beta cells. The phrase "insulin content" refers to the amount of mature insulin inside an adult beta cell. Measurement of insulin content is well known in the art. An exemplary method is extraction of cellular insulin with 3 M acetic acid. The amount of mature insulin extracted from the adult islet beta cells may be determined using an ELISA kit commercially available from Mercodia, Uppsala, Sweden.

As mentioned, the present inventors contemplate subjecting the insulin-producing cells to combinations of growth factors. Contemplated combinations include, but are not limited to IGF2 and Midkine (MDK); IGF2 and Interleukin 6; IGF2 and Pleiotrophin (Ptn); IGF2 and hCXCL2; MDK and IL-6; MDK and Ptn; MDK and hCXCL2; Interleukin 6 (IL-6) and Pleiotrophin (Ptn); Interleukin 6 (IL-6) and hCXCL2 or IGF2 and hCXCL2.

It will be appreciated that combination of at least three, four or all of the above mentioned growth factors are also contemplated.

The present inventors further contemplate culturing insulin-producing cells in a conditioned medium which comprise the above mentioned growth factors.

As used herein, the term "conditioned medium" refers to the growth medium which has been used for culturing cells for a certain culturing period. The conditioned medium includes growth factors and cytokines secreted by the cells in the culture.

According to one embodiment, the conditioned medium is generated by culturing cells (e.g. neonatal pericytes), and after an appropriate period of time, collecting the medium (i.e. isolating the medium from the cells) which includes any growth factors secreted by the cells. The cells (e.g. neonatal pericytes) for generating the conditioned medium are typically mammalian - e.g. human. The pericytes may be derived from a neonate not older than 1 month, 3 months, 6 months or even one year.

Pericytes can be identified via their expression of NG2 and isolated (by FACs) via their PDGFRbeta+CD45- signature.

The present inventors further contemplate co-culturing the insulin-producing cells of the present invention with a second population of cells, the second population of cells secreting at least one, at least two, at least three, at least four, or all of the above mentioned growth factors.

The second population of cells may be genetically modified to secrete at least one, at least two, at least three, at least four, or all of the growth factors.

Alternatively, the second population of cells naturally secretes at least one, at least two, at least three, at least four, or all of the growth factors. In a particular embodiment, the second population of cells are neonatal pericytes - as further described herein above.

Preferably, the second population of cells are readily distinguishable from the insulin- producing cells such that once sufficient quantities of insulin-producing cells have been generated, the second population of cells can be isolated from the insulin-producing cells. Methods of isolating cell populations are known in the art and include for example flow cytometry.

Since the proliferated insulin-producing cells of the present invention have the potential to store and secrete insulin, they may be used for treating a disease which is associated with insulin deficiency such as diabetes.

Thus according to an aspect of the present invention there is provided a method of treating a disease associated with insulin deficiency in a subject in need thereof, the method comprising transplanting a therapeutically effective amount of the isolated population of cells generated according to the methods described herein into the subject, thereby treating the disease associated with insulin deficiency.

Diseases associated with insulin deficiency include Diabetes.

As used herein "Diabetes" refers to a disease resulting either from an absolute deficiency of insulin (type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type 2 diabetes), i.e., impaired insulin action, in an organism. The diabetic patient thus has absolute or relative insulin deficiency, and displays, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine and excessive discharge of urine. The phrase "treating" refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition in an individual suffering from, or diagnosed with, the disease, disorder or condition. Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.

As used herein, "transplanting" refers to providing the proliferated insulin-producing cells of the present invention, using any suitable route. Typically, beta cell therapy is effected by injection using a catheter into the portal vein of the liver, although other methods of administration are envisaged.

It will be appreciated that the proliferated insulin-producing cells of the present invention can be derived from either autologous sources or from allogeneic sources such as human cadavers or donors. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene- acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagen with a ter- polymer shell of 2-hydroxyethyl methylacrylate (HEM A), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μιη. Such microcapsules can be further encapsulated with additional 2-5 μιη ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S.M. et al. Multi- layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μιη (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002;13:783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T.A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE.sup.R), etanercept, TNF. alpha, blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

The proliferated insulin-producing cells of the present invention may be transplanted to a human subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term "active ingredient" refers to the adult islet beta cells of the present invention accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer' s solution, or physiological salt buffer.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (insulin-producing cells) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., diabetes) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated from animal models (e.g. STZ diabetic mice) to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in experimental animals. The data obtained from these animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to provide cell numbers sufficient to induce normoglycemia (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.

The proliferated insulin-producing cells of the present invention may also be used for screening biologic or pharmacologic agents with B cell differentiating potential. In addition, the proliferated insulin-producing cells may be used as a source of insulin - i.e. for in vitro production. The insulin may be stored and provided directly to the diabetic patient.

As used herein the term "about" refers to ± 10 %

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

Neonatal pancreatic pericytes support beta-cell proliferation

MATERIALS AND METHODS

Mice: Mice were maintained according to protocols approved by the Institutional Animal Care and Use Committee at Tel Aviv University. All mice were maintained on a C57BL/6 background. Nkx3.2-Cre (Nkx3- 2^tml(cre)Wez) [39] mice were a generous gift. R26-YFP (Gt(ROSA)26Sor^tml(EYFP)Cos) [40] and iDTR (Gt(ROSA)26Sor^{tml(HBEGF)Awai}) [38] mice were obtained from Jackson Laboratories. Wild-type mice were purchased from Envigo, Ltd. When indicated, mice were i.p. injected with a single dose of 0.25 ng/g body weight Diphtheria Toxin (DT; List) diluted in PBS.

Islet isolation: Collagenase P (0.8 mg/ml; Roche) diluted in RPMI (Gibco) was injected through the common bile duct into the pancreas of a euthanized adult mouse. Dissected pancreatic tissue was incubated for 10-15 min at 37 °C, followed by a gradient separation with Histopaque 1119 (Sigma) for 20 min at 4 °C. Islets were collected from the gradient interface, followed by their manual collection.

Flow -cytometry: For cell sorting, dissected pancreatic tissues were digested with 0.4 mg/ml collagenase P (Roche) and 0.1 ng/ml DNase (Sigma) diluted in HBSS for 30 min at 37 °C with agitation, followed by cell filtration [41]. Cells were suspended in PBS containing 5 % FCS and 5 mM EDTA and sorted based on their yellow fluorescence by FACS Aria (BD). For staining of cell surface markers, cells were isolated as described above and stained with biotin- conjugated anti-PDGFRp (Platelet-derived Growth Factor Receptor β) antibody (Catalog #13- 1402, Affymetrix) followed by incubation with Allophycocyanin-labeled Streptavidin (Catalog #17-4317-82, Affymetrix). Cells were analyzed by a Gallios cytometer (Beckman Coulter) using Kaluza software (Beckman Coulter). For analysis of cell proliferation, single-cell suspension was obtained by incubating islets with 0.05 % Trypsin and 0.02 % EDTA solution (Biological Industries) at 37 °C for 5 min with agitation, or by collecting pTC-tet cells with 0.05 % Trypsin and 0.02 % EDTA solution (Biological Industries). Cells were fixed in 70 % ethanol at -20 °C overnight, suspended in PBS containing 1-2 % FBS and 0.09 % sodium azide, and then immunostained with Fluorescein-conjugated anti-Ki67 (Catalog #11-5698-82, eBioscience or Catalog #556026, BD) antibody. Islet cells were further stained with guinea pig anti-insulin (Catalog #A0564, Dako) antibody, followed by DyLight 650- conjugated secondary antibody (SA5-10097, Invitrogen). For analysing proliferation rates, cells were analyzed by a FACS Gallios cytometer (Beckman Coulter) using Kaluza software (Beckman Coulter). For cell counting, cells were analyzed by an Accuri C6 cytometer (BD) using its volumetric counting feature.

Cell culture: For culturing pericytes, at least 1.5 x 10⁵ sorted cells were cultured in

DMEM medium (Gibco) containing 10% FCS (Hyclone), 1% L-Glutamine (Biological Industries) and 1% Penicillin-Streptomycin solution (Biological Industries) ('complete DMEM'). Cells were sub-cultured weekly or when about 90 % confluent, using 0.25 % Trypsin solution with 0.05 % EDTA (Biological Industries). Up to their third passage, cells were plated on collagen-coated plates (Catalog #FAL354236, Corning). Media were collected from cells in their fourth passage, passed through a 22 μιη filter to exclude cells, supplemented with proteases inhibitor (Roche), and then stored at - 80 °C. Islets and pTC-tet cells were grown in complete DMEM. Growth arrest of pTC-tet cells was induced by supplementing culture medium with 1 mg/ml tetracycline (Sigma) for 10 days before a proliferation assay was performed [37,42]. For heat inactivation, pericyte-conditioned medium and complete DMEM were incubated at 62 °C for 20 min. For blocking of βΐ integrin signalling, pericyte-conditioned medium was supplemented with either hamster anti-βΐ integrin (CD29) antibody (Catalog #555003, BD) or hamster IgM (Catalog #553958, BD) as a control. Cultured pericytes were imaged using a Nikon Eclipse Ti-E epifluorescence inverted microscope.

Immunofluorescence and morphometric analyses: Dissected pancreatic tissues were fixed in 4 % paraformaldehyde for 4 h. Tissue was transferred to 30 % sucrose solution overnight at 4 °C, followed by embedding in Optimal Cutting Temperature compound (OCT, Tissue-Tek) and cryopreservation. l l^m-thick tissue sections were stained with the following primary antibodies: guinea pig anti-insulin (Catalog #A0564, Dako), rabbit anti- aSMA (a smooth muscle actin; Catalog #Ab5694, Abeam), Ki67 (Catalog #RM-9106, Thermo Scientific), and NG2 (Neural Glial antigen 2; Catalog #AB5320, Millipore),and rat anti-PECAMl (Platelet endothelial cell adhesion moleculel; Catalog #553370, BD) antibodies, followed by secondary fluorescent antibodies (AlexaFluor, Invitrogen). For TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labelling) assays, the Fluorescein In Situ Cell Death Detection Kit (Roche) was used according to manufacturer's protocol. Stained sections were mounted using Vectashield anti-fade mounting medium with DAPI (Vector). Images were acquired using an SP8 confocal microscope (Leica) or a Keyence BZ-9000 microscope (Biorevo). For analysis of islet endothelial and pericyte coverage, sections at least 50 μιη apart were stained as described. Islets were defined as insulin^"1" areas. NG2⁺ or PECAM1^"1" areas within the islets, as well as insulin^"1" areas, were measured using ImageJ software (NIH). For cell proliferation analysis, sections at least 50 μιη apart were stained as described. Images were analyzed manually blind to genotype; at least 300 insulin^"1" cells were analyzed for each pup.

Statistical analysis: Paired data were evaluated using Student's two-tailed t-test. RESULTS

Culturing neonatal pancreatic pericytes: In order to test the ability of neonatal pancreatic pericytes to promote β-cell replication in vitro, the present inventors set out to isolate and culture them. To this end, we sorted YFP-labeled cells from the pancreas of Nkx3.2- Cre;R26-YFP pups at postnatal day 5 (p5). During development, Nkx3.2 (Bapxl) is expressed in gut, stomach, and pancreatic mesenchyme, as well as in skeletal somites [43,44]. In the embryonic and adult pancreas, the Nkx3.2-Cre mouse line specifically targets mesenchymal cells, which, in the adult, consist of pericytes and vSMCs [27,33,36,41]. To determine if the Nkx3.2-Cre mouse line targets pancreatic pericytes at the neonatal age, as in adults, we analyzed fluorescently labeled ('Nkx3.2/YFPb') cells of p5 Nkx3.2-Cre;R26-YFP pancreatic tissue for PDGFR , which is expressed on the surface of pericytes but not on that of vSMCs [27]. As shown in Figure 1A, the flow-cytometry analysis revealed that approximately 90 % of Nkx3.2/YFP+ cells in the p5 pancreas express PDGFRP, displaying their pericytic identity. PDGFR -negative Nkx3.2/YFP+ cells represent vSMCs, which are targeted by the Nkx3.2-Cre mouse line but do not express this receptor [27,36]. To conclude, our results indicate that the Nkx3.2-Cre mouse line efficiently targets pericytes in the neonatal pancreas. Next, pericytes from neonatal pancreatic tissue were isolated and cultured to collect their conditioned media. To culture pericytes, dissected pancreatic tissues of p5 Nkx3.2-Cre;R26-YFP pups were digested to obtain single cells, followed by sorting of Nkx3.2/YFP+ cells by fluorescence-activated cell sorting (FACS) and culturing of sorted cells (Illustrated in Figure IB). Yellow fluorescence of cultures cells verified they were indeed sorted Nkx3.2/YFP+ pericytes (Figure 1C). Furthermore, all cultured cells extended cytoplasmic processes typical to pericytes. After four passages, the cell conditioned medium was collected and filtered to exclude cells (illustrated in Figure IB).

Pericyte-conditioned medium stimulates β-cell proliferation in vitro: To analyze the effect of neonatal pericyte-conditioned medium on β-cell proliferation in vitro, the present inventors analyzed the response of both the β-cell line PTC-tet and primary mouse adult β-cells to this medium. Immortalization of PTC-tet cells was achieved through conditional expression of SV40 (Simian Vacuolating Virus 40) large T-antigen under the control of rat Ins2 promoter [37,42]. The expression of the T-antigen under the tetracycline operon regulatory system (tet) allows for its shut-off upon exposure to tetracycline. Thus, in the presence of this antibiotic, the proliferation of PTC-tet cells becomes dependent on extrinsic factors [42,45]. To test the ability of neonatal pericyte-conditioned medium to promote proliferation of PTC-tet cells, tetracycline- treated cells were incubated with this medium. To assess the level of cell proliferation, cells were stained for the proliferative marker Ki67, and analyzed by flow-cytometry. As shown in Figure 2A, exposure to pericyte-conditioned medium promoted the proliferation of about a third of the analyzed PTC-tet cells.

Next, the ability of the pericyte-conditioned media was analyzed for its ability to promote proliferation of primary cultured adult β-cells. To this end, islets were isolated from 3-month-old mice and cultured in either control, or pericyte-conditioned medium. To assess the level of β-cell proliferation, islet cells were dispersed and stained with antibodies against insulin and Ki67, and analyzed by flow-cytometry. As shown in Figure 2B, whereas less than 1 % of β-cells cultured in control medium express Ki67, an average of 40 % of cells incubated in the presence of pericyte-conditioned medium proliferated. Next, the present inventors analyzed for a potential effect on β-cell number. As shown in Figure 2C, the number of β-cells in islets cultured for 72 hours in pericyte-conditioned medium was double the number in islets cultured in control medium. Thus, the present results indicate that factors secreted by cultured pancreatic pericytes stimulate proliferation of both a β-cell line and primary cultured adult β-cells.

Extracellular matrix (ECM) components, including these found in islets vascular BM, were shown to promote β-cell proliferation [20,28,46,47]. The present inventors thus analyzed the contribution of these factors to β-cell proliferation stimulated by pericyte-conditioned medium. First, they analyzed if proliferation of primary β-cells depends on heat- sensitive components (such as proteins) of the medium. Heating the conditioned medium (to 62 °C) prior to islet culture resulted in a low β-cell proliferation rate, which was comparable to their culturing with control media (Figure 2D). This result indicates that pericytes produce heat-sensitive factors, such as BM components and other proteins, to promote β-cell expansion. BM components are recognized by integrins and initiate downstream signaling, and integrins containing the β ΐ chain were shown to mediate β-cell proliferation [20,28]. To analyze the contribution of β ΐ integrin signaling to pericyte-mediated β-cell proliferation, βΐ integrin signaling was inhibited in βΤ^ίεί cells. To this end, the pericyte-conditioned medium was supplemented with a specific anti-β ΐ integrin blocking antibody. As shown in Figure 2E, blocking βΐ integrin signaling inhibited the expansion of βΤ^ίεί cells cultured in pericyte- conditioned medium. Of note, βΤ^ίεί cell number after their culturing in pericyte-conditioned medium supplemented with anti-β 1 integrin blocking antibody was comparable to the number of cells cultured in control medium (Figure 2E). Thus, the present analysis indicates that neonatal pancreatic pericytes stimulate β-cell proliferation in a β 1 integrin-dependent manner.

To conclude, the present analysis indicates that neonatal pancreatic pericytes secrete factors that promote β-cell proliferation.

Diptheria toxin-mediated depletion of neonatal pancreatic pericytes: To analyze the in vivo role of neonatal pancreatic pericytes, this cell population was depleted using the Diphtheria Toxin Receptor (DTR) system. To deplete pericytes, Mx?.2-Cre;iDTR mice were generated, which express DTR in a Cre-dependent manner [36]. Cell-specific expression of the iDTR transgene, combined with DT administration, serves as a tool for targeted cell ablation [48,49]. To deplete pericytes in neonatal pancreas, N£x?.2-Cre;iDTR pups as well as control littermates (iDTR-transgenic not expressing the Nkx3.2-Cre) at p3 were i.p. injected with DT (Figure 3A). In addition to its pancreatic expression, the x?.2-Cre line also displays non-pancreatic expression in the joints and gastro-intestinal mesenchyme [39,50]. Treating neonatal mice with the DT dose used for treating adult mice (4 ng/gr body weight [36]) attenuated the growth and survival of N£x?.2-Cre;iDTR transgenic pups. Therefore, the dose of injected DT was titered to ensure that the growth of the pups would be unaffected by the treatment. The results indicated that injecting p3 Mx?.2-Cre;iDTR pups with 0.25 ng/gr body weight DT allowed them to grow normally, as manifested by a body weight comparable to their control littermates at ages p5 and p21 (Figure 3B), and their long-term survival. This indicates that weight gain and growth were unaffected in DT-treated x?.2-Cre;iDTR pups.

To assess pericyte depletion, islet pericyte and endothelial coverage was measured for pups at p5. The morphometric analysis revealed that DT treatment of x?.2-Cre;iDTR pups led to -25% reduction in islet pericyte coverage, identified by the expression of the pericytic marker NG2 (Figures 3C and D). In contrast, islet coverage by endothelial cells, identified by the expression of PECAM1, remained unchanged (Figures 3C and E). Notably, the present inventors did not observe gross changes in the coverage of large pancreatic vessels by vSMCs (identified by the expression of high aSMA levels; Figure 3F) in DT-treated Mx?.2-Cre;iDTR pups as compared to control. Thus, treatment of x?.2-Cre;iDTR pups with a low DT dose allows partial, but specific, depletion of their islet pericytes without affecting their growth.

Depletion of neonatal pancreatic pericytes impairs β-cell proliferation in vivo: To determine whether neonatal pancreatic pericytes are required for β-cell proliferation, pancreatic pericytes were depleted by treating N£x?.2-Cre;iDTR pups with DT. Determining primary, rather than secondary effects requires studying short-term events. Therefore, Mx?.2-Cre;iDTR pups and control (iDTR-transgenic not expressing the x?.2-Cre) littermates were treated with 0.25 ng/gr body weight DT at p3 and analyzed 2 days after DT administration, at p5. To analyze the effect of pericyte depletion on β-cell proliferation, the percentage of Ki67⁺ cells were measured out of the total number of insulin-expressing cells in pancreatic tissues of DT-treated Nkx3.2- Cre;iDTR and control pups by immunofluorescence (Figure 4A). This morphometric analysis indicated a significant reduction of the portion of proliferating β-cells in DT-treated Nkx3.2- Cre;iDTR mice to about two thirds of that observed in littermate controls (Figure 4A).

The observed reduced β-cell proliferation may result from the impaired survival of these cells. The present inventors therefore performed TUNEL assays on pancreatic tissue sections from DT-treated Mx?.2-Cre;iDTR and control p5 pups to analyze for potential β-cell apoptosis. This analysis did not indicate β-cell death upon pericyte depletion (Figure 4B).

To conclude, the results indicate that reduced pericyte density impairs β-cell proliferation in vivo, indicating that pancreatic pericytes are required for neonatal β-cell expansion.

EXAMPLE 2

Growth factors secreted by neonatal pancreatic pericytes support beta cell proliferation

To identify pericytic growth factors with an age-dependent expression pattern, the present inventors compared pancreatic pericytes isolated from pO and p5 (both associated with high β- cell replication rate; Figure 5) as well as pl4 and p21 (associates with low β-cell replication rate; Figure 5). RNAseq analysis yielded 782 genes with a significantly higher expression level in pericytes isolated from p5 pancreata as compared to these isolated from p21. Gene Ontology (GO) analysis revealed that five of those genes encode proteins with a predicted growth factor activity: Insulin Growth Factor 2 (IGF2), Midkine (MDK), Interleukin 6 (IL-6), Pleiotrophin (PTN) and Cxcll (the mouse ortholog of human CXCL2)(Figure 5). Of note, the expression pattern of these genes closely mimics that of age-dependent β-cell replication rate; i.e., high expression levels during periods of extensive β-cell proliferation (pO and p5), and low-to-no expression in ages in which β-cell proliferation rate is low (p21). To determine the functionality of these factors, the present inventors analysed the response of the mouse β-cell line βΤ^ίεί, in which cell proliferation becomes dependent on extrinsic factors in the presence of tetracycline , to two of the five identified pericytic factors (in the form of recombinant proteins). As shown in Figure 6, both IGF2 and PTN promoted β-cell proliferation.

The work leading to this invention has received funding from the European Research Council under the European Union's Seventh Framework Programme ERC-2013-StG/ERC grant agreement no. 336204. REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. An in vitro method of promoting proliferation of insulin-producing cells, the method comprising subjecting insulin-producing cells to an effective amount of at least one growth factor expressed by pancreatic pericytes at the neonatal period under conditions which allow the proliferation of the said insulin secreting cells by at least two fold, with the proviso that said at least one growth factor is not insulin-like growth factor 2 (IGF2), thereby promoting proliferation of insulin-producing cells.

2. An in vitro method of promoting proliferation of insulin-producing cells, the method comprising subjecting insulin-producing cells to an effective amount of at least two growth factors expressed by pancreatic pericytes at the neonatal period, thereby promoting proliferation of insulin-producing cells.

3. The method of claim 1, wherein said at least one growth factor is selected from the group consisting of Midkine (MDK), Interleukin 6 (IL-6), Pleiotrophin (Ptn) and human CXCL2.

4. The method of claim 2, wherein said at least two growth factors are selected from the group consisting of IGF2, Midkine (MDK), Interleukin 6 (IL-6), Pleiotrophin (Ptn) and human CXCL2.

5. The method of any one of claims 1-4, wherein said growth factor is a recombinant growth factor.

6. The method of any one of claims 1-5, wherein said subjecting is effected by culturing the cells in the presence of said growth factor or a functional equivalent thereof.

7. The method of any one of claims 1-4, wherein said subjecting is effected by culturing the cells in a conditioned medium comprising said growth factor.

8. The method of claim 7, wherein said conditioned medium is generated by culturing neonatal pericytes.

9. The method of any one of claims 6-8, wherein said culturing is effected for at least 10 passages.

10. The method of any one of claims 1-4, wherein said subjecting is effected by co- culturing the cells with an additional population of cells that secrete said growth factor.

11. The method of claim 10, wherein said additional population of cells comprises neonatal pericytes.

12. The method of any one of claims 1-4, wherein said subjecting is effected by expressing in the cells said growth factor or a functional equivalent thereof.

13. The method of any one of claims 1-12, wherein said growth factor comprises at least 2 and no more than 5 growth factors.

14. The method of claim 1, wherein said at least one growth factor comprises MDK and IL-6; MDK and Ptn; MDK and human CXCL2; Interleukin 6 (IL-6) and Pleiotrophin (Ptn); or Interleukin 6 (IL-6) and human CXCL2.

15. The method of claim 2, wherein said at least two growth factors comprise IGF2 and Midkine (MDK); IGF2 and Interleukin 6; IGF2 and Pleiotrophin (Ptn); IGF2 and human CXCL2; MDK and IL-6; MDK and Ptn; MDK and human CXCL2; Interleukin 6 (IL-6) and Pleiotrophin (Ptn); Interleukin 6 (IL-6) and human CXCL2 or IGF2 and human CXCL2.

16. A method of promoting proliferation of insulin-producing cells, the method comprising culturing the insulin-producing cells in a conditioned medium of neonatal pericytes, thereby promoting proliferation of insulin-producing cells.

17. The method of any one of claims 1-16, wherein said insulin-producing cells are pancreatic beta cells.

18. The method of claim 17, wherein said pancreatic beta cells are cadaveric cells.

19. The method of any one of claims 1-16, wherein said insulin-producing cells are stem cell-derived.

20. The method of any one of claims 1-17, wherein said insulin-producing cells are adult cells.

21. The method of any one of claims 1-17, wherein said insulin-producing cells are fetal cells.

22. The method of any one of claims 1-17, wherein said insulin-producing cells are human cells.

23. In vitro expanded cells obtainable according to the method of any one of claims

1-22.

24. Use of the cells of claim 23 in the manufacture of a medicament for treating insulin deficiency.

25. The use of claim 24, wherein said insulin deficiency is associated with TIDM or

T2DM.

26. A method of generating a conditioned medium useful for proliferation of insulin- producing cells the method comprising:

(a) culturing neonatal pericytes in a medium for a predetermined time so as to generate a medium comprising growth factors secreted from said neonatal pericytes; and

(b) isolating said medium comprising growth factors from said neonatal pericytes, thereby generating the conditioned medium.