WO1997016536A1

WO1997016536A1 - Method for ex vivo proliferation and differentiation of adult pancreatic islet cells, media useful therefor and uses thereof

Info

Publication number: WO1997016536A1
Application number: PCT/US1996/016396
Authority: WO
Inventors: Patrick Soon-Shiong; Maria Varsanyi-Nagy; Kevin Ferreri; Molly Moloney; Roswitha Heintz
Original assignee: Vivorx, Inc.
Priority date: 1995-10-30
Filing date: 1996-10-11
Publication date: 1997-05-09
Also published as: AU7443996A

Abstract

A method for inducing the profileration and differentiation of neonatal and/or adult human or non-human pancreatic islets to produce a product useful, for exmple, as a therapeutic agent for treatment of diabetes has been developed. Invention method involves a series of complex cell culture media containing necessary nutrients and growth factors, a human cytokine (hepatocyte growth factor or scatter factor), a microgravity culture vessel for promoting three dimensional growth, and molecular biology assays for measuring insulin promoter activity. A method for providing a hybrid organoid comprising a combination of donor and recipient cell types is also described.

Description

Method for Ex Vivo Proliferation and Differentiation of Adult Pancreatic Islet Cells.

Media Useful Therefor and Uses Thereof

Field of the Invention

The present invention relates to methods and compositions for the proliferation of adult human or non-human pancreatic islets of Langerhans. Invention methods are useful, for example, to provide a therapeutic for treatment of type 1 diabetes mellitus, wherein such cells are optionally encapsulated within alginate microcapsules before delivery to a subject. The invention employs a series of complex cell culture media containing various nutrients which are sufficient to promote long term cell growth or multiplication and to avoid senescence or loss of biological function. The invention includes methods for the proliferation of islets in the form of high fidelity three dimensional tissue-like structures employing a microgravity culture vessel. In another aspect, the invention utilizes a novel beta cell marker using molecular biology for specifically measuring the transcriptional activity of the insulin promoter in pancreatic beta cells. In yet another aspect, the invention provides a combination of donor and recipient cell types to provide an organoid with reduced immunogenic potential.

Background of the Invention

Type 1 insulin dependent diabetes mellitus is characterized by the loss of insulin producing beta cells from the pancreatic islets of Langerhans. Standard therapy has included parenteral administration of insulin (either bovine or porcine or recombinant human) by means of multiple daily injections or an indwelling catheter and pump, but this treatment can only temporarily delay the pathological complications of the disease. Adult human pancreatic islets have been transplanted into patients to achieve independence from insulin injections. Such transplantations require the use of immunosuppressive treatment (e.g., with cyclosporine A) to prevent rejection of the transplanted cells. The use of immunosuppressive treatment has been limited, however, by toxic side effects and by increased potential for infection. Moreover, inadequate supplies of human islets and the complications of graft rejection have necessitated the search for an improved source of islet cells.

Fetal pancreatic islets contain many undifferentiated beta cells which can mature after transplantation and which are less subject to rejection by the recipient, but they cannot be obtained in large enough quantities to serve as a practical therapeutic approach. Transplantation of individual cells or cellular communities (including human or porcine pancreatic islets, hepatocytes, keratinocytes, chondrocytes, acinar cells, or chromaffin cells) will require a virtually inexhaustible supply of functional living cells for use in human therapy.

While it has been reported that proliferation may occur from fetal tissue, the ability to proliferate adult, terminally differentiated islet cells, has eluded investigators. Each mature islet of Langherhans is a cellular community comprising four distinct cell types arranged in the typical topographical distribution. At the central core are the beta cells, which secrete insulin in response to elevated glucose. At the outer peripheral rim are alpha cells (which secrete glucagon) , PP cells (which secrete pancreatic polypeptide) , and Delta cells (which secrete somatostain) .

The ability to demonstrate ongoing physiological release of insulin following multifold proliferation of fully differentiated adult islet cells has eluded investigators in the past. Attempts by previous investigators have failed due to overgrowth of the beta cells by fibroblasts, resulting in negative selection of endocrine secreting cells, and thus cessation of insulin secretion from the proliferated cells.

Accordingly, there is a clear need in the art for methods to promote the proliferation of fully differentiated adult islet cells to produce increased quantities of cells which are capable of ongoing physiological release of insulin.

Proliferation of mammalian cells presents special problems. Thus, in contrast to mammalian cells, free living microorganisms (e.g., bacteria or fungi) possess tough cell walls for resistance to most environmental stresses. In addition, such organisms synthesize nearly all of the biomolecules essential to life. However, cells from higher organisms (e.g., mammals or humans) are structurally delicate and require constant supplements of specific nutrients to maintain viability. Large scale processes are better developed and less difficult for culturing bacteria than for culturing mammalian cells. Thus, while bacterial cells can be grown under vigorous agitation in large volumes of simple liquid medium, mammalian cells, in contrast, are more difficult to grow: they are easily damaged by the shear stresses of turbulent fluid flow, they require complex nutrient media to support cell growth, and they often grow better in the presence of an appropriate substrate surface which promotes cell attachment.

As an alternative to proliferation of mature adult cells, immortal carcinoma cell lines have been developed and propagated for many years. Prior art techniques, however, have not permitted the growth of non- neoplastic, non-transformed cells (including adult pancreatic islets) in large scale three dimensional cultures (which promote the important cell-to-cell contacts found in natural tissue) . While small scale culture systems (e.g., plastic plates with microwells) are adequate for laboratory experiments, they do not provide enough surface area for commercial production. Certain carcinomas have been successfully grown in the presence of a pre-established stromal support matrix, which is composed of non-living material and inoculated with stromal cells. Free floating tumor cell spheroid aggregates, both with and without attachment substrates such as microcarriers, have provided material for experimental analysis of embryological development and chemotherapeutic cytotoxicity. Collagen coated cellulose sponges have allowed carcinoma cells to adhere, migrate, and proliferate on a solid substrate in the presence of fibrin, although degenerative changes were detected after ten days of culture. Microcarrier beads provide increased surface area for cellular attachment, allowing them to assemble into tissue-like three dimensional structures which mimic the natural relationships. Agitation in a conventional impeller driven bioreactor vessel suspends the cells in the medium, delivers fresh nutrients, and removes metabolic waste products, but it also subjects them to high levels of shear stresses which can damage cells and inhibit cellular tissue assembly.

Accordingly, what is needed in the art are methods to promote the proliferation of fully differentiated adult islet cells to produce increased quantities of cells which are capable of ongoing physiological release of insulin. Especially desirable would be methods for the production of such cells under conditions which promote assembly into tissue-like three dimensional structures which mimic the natural relationships of cells in native tissues. Brief Description of the Invention

In one aspect of the present invention, it has been discovered that a specifically defined media, as well as a specifically defined environment in which the cells are cultured, allow proliferation of adult differentiated islet cells, with ongoing insulin secretion in the cells thus proliferated.

In accordance with another aspect of the present invention, there has been developed a method of proliferating human and non-human islet cells to produce huge quantities of synthetic islet cells which demonstrate continued physiological activity. The invention method is made possible by the identification of specific defined cell culture media, growth factors, differentiation factors and environmental factors which enhance the selection of beta cells. With this accomplishment, it is now possible to provide a virtually unlimited supply of insulin- secreting cells for the treatment of insulin dependent diabetic patients.

In accordance with yet another embodiment of the present invention, it has been discovered that by surrounding the cells in a physical environment which mimics that of the fetus in the pregnant uterus (i.e., by suspending the cells in a cell culture device which allows low shear as well as maximum co-localization of the cells) , maximum pseudo islet (i.e., islet-like) or aggregate formation is induced, resulting in the formation of organoids with excellent secretory function and differentiation.

Having developed means to produce proliferated single beta cells, or endocrine cells, from islets, there have further been developed means whereby these single islet cells can be reaggregated into a three dimensional morphology resembling that of natural intact islets containing alpha, beta, delta, and PP cells. These pseudo islets are reaggregated under conditions which appear to further enhance their state of differentiation. The conditions under which this is accomplished is one of low shear using a device which provides for microgravity conditions.

The invention also describes a method of co-culturing proliferated islets from a donor pancreas, together with cell types optionally obtained from the recipient. These cell types include fibroblast, endothelial neural cells, and the like, which together may reduce the immunogenicity of the hybrid co-cultured organoid, as well as enhance the functional activity of the resulting pseudo islet. In addition, it has also been discovered that co-culturing freshly isolated, non- proliferated islets with islet cells which have undergone proliferation, and encapsulating these co-cultured organoids, results in cells having a longer viability, stability and insulin secretory activity than do either of the components of the co-cultured organoid alone. Furthermore, there is also the potential of encapsulating, together with these proliferated islets, other cell types (including acinar cells, hepatocytes, and the like) which may provide enhanced activity of these co-cultured hybrid organoids.

Further in accordance with the present invention, the somatostatin transcription factor, STF-l, has been used as an identifying marker to detect specific functional activity of the insulin promoter concurrent with insulin expression in the beta cells of pancreatic islets. This STF-l factor is used as a probe, not only to optimize the cell culture media in terms of developing a media which provides the highest STF-l activity, but also provides a probe for identifying STF-l cells, and thus insulin secreting cells. Immunohistochemical and molecular biological techniques involving antibodies to STF-l make it possible to monitor and analyze insulin expression at the level of transcription of DNA into mRNA within the cell nucleus.

Brief Description of the Figures

Figure 1 presents a flow chart outline of the invention method whereby adult human islet cells are produced by initiation, expansion and termination of adult human islet cell proliferation and pseudo islet formation in vitro.

Figure 2 presents a growth curve for proliferating human adult islet cells.

Figure 3 presents a diagram of the population doubling time of human adult islet cells generated by cell counting. About 20 doublings are required to produce 1 million cells.

Figure 4 presents a comparative bargraph representing the glucose + theophylline stimulated insulin secretion from 12,000-fold expanded islet cells after 24 h aggregation and encapsulation.

Figure 5A represents the perifusion curve of normal adult islets.

Figure 5B is a graphic presentation of the insulin secretion rate of 12,000-fold expanded adult human pseudo islets after encapsulation.

Figure 6A is a graphic presentation of insulin secretion from co-encapsulated adult islets with 1000-fold expanded pseudo islets. Figure 6B is a graphic presentation of insulin secretion from encapsulated proliferated islets alone. Comparison of Figures 6A and 6B demonstrate enhanced viability and function of co-cultured islets.

Detailed Description of the Invention

In accordance with the present invention, there is provided a method for stimulating the ex vivo proliferation and differentiation of neonatal and/or adult pancreatic islet beta cells, said method comprising: (a) contacting a primary culture of neonatal and/or adult pancreatic cells under conditions suitable to induce beta cell proliferation; and

(b) contacting the differentiated cells produced in step (a) under conditions suitable to induce prolonged proliferation of said cells.

As employed herein, "primary culture" refers to a mixed cell population of neonatal and/or adult pancreatic cells which permits interaction of epithelial and mesenchymal cells within islet-like cell clusters.

As employed herein, "ex vivo" refers to cells which have been taken from a body, temporarily cultured in vivo , and then returned to a body.

As employed herein, "proliferation" refers to an increase in cell number.

As employed herein, "differentiation" refers to increased numbers of islet-like cell clusters containing an increased proportion of beta epithelial cells which produce increased amounts of insulin per cell.

Pancreatic tissue source suitable for use in the practice of the present invention include adult human pancreases (which can be obtained from cadaver organ donors, aε well as living donors) , neonatal human pancreases, neonatal and/or adult porcine pancreases, and the like. Non-human adult pancreata are obtainable from porcine or bovine sources. Pancreata are typically shipped on ice in standard medium (e.g., RPMI 1640, Irvine Scientific, Irvine, CA) supplemented with 10% normal human serum and antibiotics (penicillin 100 U/ml, streptomycin 0.1 mg/ml, and amphotericin B 1 mg/ml), and are processed within 6 to 12 hours of retrieval.

Pancreatic islet isolation can be carried out as known in the art, for example, by digesting human and non¬ human adult pancreata using collagenase (e.g., collagenase P, Boehringer, Indianapolis, IN) under aseptic conditions (see, for example, Soon-Shiong et al., in Transpl . 54:769- 774 (1992)). Islets are purified by gradient technical separation, viability tested by acridine orange/propidium iodide uptake, and beta cell concentration estimated via a specific vital dye stain (dithazone) i.e., DTZ uptake.

Explants subjected to tissue culture in accordance with the present invention may consist of highly purified adult islets (human or porcine) , or of adult islets mixed with exocrine or duct tissue, or of disaggregated single cells of highly purified adult islets.

Islet cell proliferation is initiated from highly purified whole islets, instead of monodispersed islet cells. The advantage of not starting with single cells can be explained with reference to both the physiology and anatomy of the islet microorganε (see, for example, Pipeleers et al., in Proc. Natl . Acad . Sci . USA 79:7322- 7325 (1982) , Cell Biology Section) . Glucose-induced insulin release depends on functional cooperation between islet cells. Exposure to glucose caused release of 30-fold more insulin from beta cells lodged within intact islets as from purified single beta cells. Structurally coupled beta cells and single beta cells isolated with alpha cells responded 4-fold more effectively to glucose than single beta cells. Glucose responsiveness is dependent not only the number and integrity of insulin containing beta cells, but also their interactions with their neighboring beta and non-beta cells. Insulin secretion is seen to depend on the micro anatomy and functional organization of the islets. (Pipeleers et al., supra) .

The solid growth substrate may be a surface- treated polystyrene petri dish (FALCON 3003) , a tissue culture flask, or the like, coated with a variety of agents (e.g., Matrigel, laminin, fibronectin, collagen, hyaluronic acid, and the like) for selective attachment. These solid growth substrates may be re-used after trypsinization. Islet cells may be co-cultured with fibroblast cells. The differentiated state is induced by extracellular matrix, by growth factors, or by contact with neighboring cells. The differentiated state is stabilized by cell-cell adhesion, cell-cell communication, cell substrate adhesion, cell substrate interaction, and the like.

Various nutrients and factors supplemented into the growth medium are critical for the long term viability and health of mammalian cells in culture. In accordance with the present invention, a combination of ingredients in appropriate amounts has been demonstrated to enhance the proliferation of adult pancreatic islets, thereby providing a virtually unlimited supply of therapeutic material, exceeding the supply available from natural sources. In order to induce differentiated adult insulin secreting cells to proliferate and maintain their physiological status, it has been discovered that a molecular environment simulating that of the pregnant state must be provided to the cells in culture. This molecular environment includes factors which 1) induce specific growth of beta cells (scatter factor) , 2) induce cellular growth and mitosis, 3) promote differentiation (nicotinamide) , and 4) inhibit fibroblast overgrowth. All of these factors, in combination, are critical to achieving proliferated beta cells which are capable of insulin secretion following multiple fold mitosis.

Suitable culture medium is prepared using a standard commercially available cell culture medium (e.g., RPMI DMEM, Ham's F12) as a base, at a pH of about 7.4, containing an effective cell growth promoting concentration of water, calcium ions, sodium ions, glucose, insulin, transferrin, all essential amino acids, water soluble vitamins, coenzymes, and glucose. The culture medium should contain a source of an aqueous mixture of lipoprotein, cholesterol, phospholipids, and fatty acids with low endotoxin. A broad spectrum antibiotic (e.g., gentamicin) can also optionally be included in the culture medium to prevent contamination by bacteria, yeast, or fungi.

Hepatocyte growth factor is added to the culture medium to stimulate the proliferation of adult pancreatic beta cells from primary cultures of adult pancreatic cells, to increase insulin production in primary and secondary cultures of adult pancreatic islet cells, and to prepare large quantities of functional adult pancreatic beta containing islets for transplantation into diabetic patients.

Hepatocyte growth factor (HGF or scatter factor) is an 87 kDa two chain glycoprotein cytokine, a potent hepatocyte mitogen, and a fibroblast secretory protein which increases the motility of epithelial cells. It has been purified to homogeneity, sequenced, and genetically cloned. It was identified immunohistochemically in pancreatic glucagon secreting A cells (but not exocrine cells) of adult humans or rats, and also in developing pancreatic acinar cells of fetal rats.

In accordance with the present invention, nicotinamide is added to cell growth media at the appropriate concentrations and at the appropriate stages of the proliferation, thereby inducing specific beta cell differentiation. While nicotinamide has been discovered to be toxic to cells when present at too high a concentration, it has also been discovered that the presence of nicotinamide at the appropriate stages of the culture period serves not only to desirably inhibit fibroblast overgrowth, but also to desirably induce beta cell differentiation and increase insulin content and output.

In addition, in accordance with the present invention, it has been discovered that in order to induce proliferation in adult differentiated cells, it is important to provide hormones which mimic the pregnant state. These hormones include human placental lactogen, hormones of the pituitary (including corticotropin, somatotropin, oxytocin, vasopressin, and the like) , as well as hormones provided from hypothalamic extracts (e.g., growth hormone releasing hormone, thyrotropin releasing hormone, corticotrophin releasing hormone, gonadotropin releasing hormone, luteinizing releasing hormone, prolactin releasing hormone, adrenocorticotropic hormone, thyrotropin stimulating hormone, follicle stimulating hormone, luteinizing hormone, and the like) . The presence of these hormones in the mileau during the proliferation phase have been shown to induce proliferation of terminally differentiated cells. In the absence of these factors, a significant reduction, and even absence, of growth in these differentiated cells is observed. An important aspect of this invention is the discovery of methods which simultaneously inhibit fibroblast overgrowth, while at the same time specifically induce (through the addition of the hormones and growth factors described above) selection and proliferation of endocrine secreted cells. Fibroblast inhibition is accomplished by significantly lowering the serum concentration, as well as by the addition of nicotaminide.

Thus, growth media contemplated for use in the practice of the present invention are established at a pH of 7.4, an osmolarity between 270 and 320 mOsmol, a temperature of 37°C, and surface tension sufficiently low to prevent formation of air bubbles. The media contain an effective cell growth promoting concentration of water, sodium ions (Na⁺) , potassium ions (K⁺, 0.23 g/L), calcium ions (Ca⁺⁺, between 0.37 and 1.1 mM) , magnesium ions (Mg⁺⁺) , zinc ions (Zn⁺⁺) , chloride ions (Cl ) , sulfate ions (S0₄ ) , bicarbonate ions (HC0₃ ) , glucose (1500 mg/L) , all essential amino acids, cysteine, tyrosine, glutamine (between 2 and 7 mM) , water soluble vitamins, nicotinamide, coenzymes, and inorganic trace elements. Glucose is preferably present at 0.8 to 1.2 mg/mL. The culture medium contains a source of an aqueous mixture of lipoprotein, cholesterol, phospholipids, and fatty acids with low endotoxin. A broad spectrum antibiotic (e.g., gentamicin) can optionally be included in the culture medium to prevent contamination by bacteria, yeast, or fungi. The media described in the present invention are of various types for use at different stages of the proliferation and differentiation process. They include:

I. VRX-E: Establishing media for endocrine cell selection (see Example 1) . II. VRX-S: Beta cell specific differentiation media containing various concentrations of nicotinamide (see Example 2) .

III. VRX-P: Extended proliferation media for extended propagation. This medium contains a reduced dose of nicotinamide, and optionally no scatter factor. IV. VRX-C: Media to bring about cessation of proliferation whereby growth factors are removed from the base medium.

V. VRX-A: Media utilized for the aggregation of pseudo islets.

Growth factors, hormones and differentiation factors contemplated for use in the above-described media include pregnancy hormones (e.g., lactogen) , gastrointestinal hormones (e.g., gastrin or CCK) , pituitary hormones (e.g., prolactin or growth hormone), steroid hormones, thyroid hormones (e.g. T₃ or T₄) , insulin (as Na insulin monomer) , epidermal growth factor (EGF) , hepatocyte growth factor (HGF) , fetal bovine serum (FBS) 4%, attachment factors, spreading factors, binding proteins, and the like.

Gas phase employed for the above-described culturing is introduced as follows: The culture is perfused with a gas mixture comprising either 5% C0₂ plus 95% air plus 2.5 ng/mL selenous acid in a C0₂ incubator, or 95% 0₂ plus 5% N₂. The gas temperature is maintained at 37°C and the relative humidity is maintained at 90%.

Optionally, the above-described method further comprises:

(c) contacting the proliferated cells produced in step (b) under conditions suitable to arrest the growth thereof, and thereafter, optionally

(d) culturing the cells produced in step (c) under conditions suitable to promote the formation of three dimensional tissue-like structures.

Conditions suitable to arrest the growth of said cells comprise culturing the differentiated/proliferated cells in growth cessation media, VRX-C. Conditions suitable to promote the formation of three dimensional tissue-like structures comprise culturing cells in a microgravity device in the presence of aggregation media, VRX-A. Thus, in accordance with the present invention, it has been discovered that the microgravitational process greatly enhances the capability to form and maintain three dimensional islet clusters. It has been discovered that by using a rotational wall vessel (such as, for example, the device disclosed by Schwarz et al. , in U.S. Patent No. 4,988,623, or the device disclosed by Schwarz et al., in U.S. Patent No. 5,026,650, both of which are hereby incorporated by reference herein in their entirety) , the low gravity process simultaneously minimizes the fluid shear stress, provides three dimensional freedom for islet cell cluster and substrate spatial orientation, and increases localization of the various cell types of the islet (i.e. Delta, Beta and PP cells) in a similar spatial region for significant periods during the cell culture, thereby increasing the pseudo islet formation. Cells and substrate rotate about an axis nearly perpendicular to gravity. Cells of greatly different sedimentation rates orbit in particular paths and remain spatially localized for many minutes or hours. This allows individual islet cells sufficient interaction time to form multicellular structures and to associate with each other. A vessel diameter is chosen which has the appropriate volume for the intended quantity of cultured material and which will allow a sufficient seeding density of cells, tissues, and substrates. The outward particle drift due to centrifugal force is exaggerated at higher vessel radii and for rapidly sedimenting particles. Selected levels of shear stress may be introduced into the culture environment by differential rotation of the vessel components, as a means for controlling the rate and size of tissue formation and for maintaining optimal particle sizes and associated sedimentation rates. Individual pancreatic islet cells cultured under microgravity conditions lead to the formation and maintenance of three dimensional aggregates possessing similar morphology and anatomical structure to that normally found in natural tissue. Thus, islet cells (after several fold expansion) were introduced into a microgravity vessel containing culture medium, growth factors, and an attachment matrix. Simulated microgravity was created (in ordinary unit gravity) by modulating the horizontal rotation of a culture vessel completely filled with culture medium containing the matrix. These conditions cause cells to co-locate in one spatial region and encourage the maintenance of aggregates because shear stresses arising from the relative motion of the medium with respect to the walls of the vessel are minimized.

Utilizing the above-described microgravity vessel technique, it has also been discovered that it is possible to co-culture proliferated islet cells, together with cells of other types (e.g., freshly isolated islets, endothelial cells, acinar cells, neural cells and cells from other solid organs such as hepatocytes, and the like) . Enhanced activity of cells co-cultured in this manner has been demonstrated. The advantages of such co-cultured cells may be in their reduced immunogenic profile, as well as in their enhanced differentiated state, resulting in an enhanced secretion of the desired hormone or peptide.

In accordance with another embodiment of the present invention, there is provided a method for treating a subject with type 1 diabetes mellitus, said method comprising:

(a) contacting a primary culture of neonatal and/or adult pancreatic cells under conditions suitable to induce beta cell proliferation; (b) contacting the differentiated cells produced in step (a) under conditions suitable to induce prolonged proliferation of said cells;

(c) contacting the proliferated cells produced in step (b) under conditions suitable to arrest the growth thereof;

(d) culturing the cells produced in step (c) under conditions suitable to promote the formation of three dimensional tissue-like structures containing increased numbers of insulin producing islet-like cell clusters containing beta epithelial cells;

(e) encapsulating said cells or islet-like cell clusters; and

(f) parenterally administering an effective amount of said encapsulated cells to said subject.

In accordance with yet another embodiment of the present invention, there is provided a method to proliferate or differentiate neonatal and/or adult pancreatic islet cells in clinically useful quantities, said method comprising:

(a) seeding a primary culture of neonatal and/or adult pancreatic cells into a microgravity vessel;

(b) contacting said cells in said microgravity vessel with a complete growth medium supplemented with a proliferation inducing amount of a cytokine (hepatocyte growth factor) for a time sufficient to allow differentiation, proliferation and aggregation of said cells; and thereafter

(c) harvesting the resulting islet-like cell clusters containing beta epithelial cells from said microgravity vessel; and optionally

(d) encapsulating said islet-like cell clusters.

Encapsulation is optional because of the low immunogenicity of cells prepared as described herein. In accordance with still another embodiment of the present invention, there is provided a composition for transplanting functional neonatal and/or adult pancreatic tissue into patients, said composition comprising: a pharmaceutically acceptable vehicle, containing primary cultures of neonatal and/or adult pancreatic islet cells which have been contacted ex vivo with a differentiation and proliferation inducing amount of a cytokine (hepatocyte growth factor) sufficient to induce an increase in cell number, an increase in the formation of islet-like cell clusters containing beta epithelial cells, and retain the ability to produce insulin in response to stimulus.

Exemplary pharmaceutically acceptable vehicles include alginate microcapsules, as well as sterile aqueous or non-aqueous solutions, suspensions, or emulsions.

Examples of non-aqueous vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.

In accordance with a still further embodiment of the present invention, there is provided a composition comprising a combination of freshly isolated islet cells and proliferated neonatal and/or adult islets. Such compositions are optionally encapsulated to facilitate administration to a patient. Thus, for example, such compositions can be employed for treating a subject with type 1 diabetes mellitus. This is accomplished by parenterally administering an effective amount of the above-described optionally encapsulated combination of cells to said subject.

In accordance with a further embodiment of the present invention, there are provided methods to identify proliferated and/or differentiated cells as beta cells, said method comprising monitoring said cells for the expression of STF-l. Such method can also be employed to optimize culture media for the differentiation of epithelial cells for selection for insulin-secreting beta cells. In this aspect, a cell population is monitored for the presence of STF-l as a function of variations in the culture media, and the media then modified so as to maximize expression of STF-l.

Immunohistochemistry and immunocytochemistry:

Phenotyping of proliferating cells is accomplished using immunochemical techniques directed against insulin, glucagon, somatostatin, SF receptor, STF-l, vimentin, and Factor VIII.

Evaluation of differentiated, proliferated islets: The polymerase chain reaction (PCR) can be used to phenotype proliferated cells. Function in vitro is assessed by sterile static glucose stimulation of adherent cells and by dynamic glucose stimulation to modulate the insulin secretion rate. Cell aggregation and pseudo islet formation is performed in the microgravitation system. Function in vivo is assessed by transplantation of encapsulated islet cell aggregates into mice with autoimmune diabetes.

Somatostatin transcription factor (STF-l) regulates insulin expression in beta cells of pancreatic islets. It stimulates the insulin gene by recognizing two islet specifying elements on the insulin promoter. Specification of the four islet cell types (secreting either insulin, glucagon, somatostatin, or pancreatic polypeptide) during development may be partially determined by the expression of STF-l relative to other islet cell factors, since the development of endocrine cell types in the pancreas is believed to involve the progressive restriction of pluripotent stem cells.

STF-l is a member of the homeobox class of transcription factors and is required for pancreatic organogenesis (Jonsson et al., Nature 371:606-609 (1994)). It is expressed during early development by the epithelial cells of the gut and most of the cells that will eventually form the pancreas. However, in the adult, STF-l expression is lost in pancreatic ductal, exocrine, and alpha cells, and is restricted to the duodenal epithelium, beta cells, and a subset of delta cells (Guz et al., Development 121:11-18 (1995)). In adult beta and delta cells, STF-l is required for the hallmark phenotype of these cells, the expression of insulin in beta cells (Peers et al., Mol Endocrinol 8:1798-1806 (1994)) and somatostatin in delta cells (Leonard et al., Mol Endocrinol 7:1275-1283 (1993)). STF-l binds to the CT2 box in the human insulin promoter (Petersen et al., Proc Nat Acad Sci USA 91:10465-10469 (1994)) resulting in increased transcription of the insulin gene. In accordance with the present invention, STF-l is utilized as both a marker for mature islet cells and as a requirement for insulin expression. Further in accordance with the present invention, culture conditions are optimized based upon STF-l expression and activity in conjunction with measurements of glucose responsive insulin release.

In accordance with a still further aspect of the present invention, there is provided a method for the preparation of cell clusters from proliferated, differentiated, growth arrested cells, said method comprising subjecting said cells to aggregation conditions in a microgravity vessel, such as, for example, the device disclosed by Schwarz et al., in U.S. Patent No. 4,988,623, or the device disclosed by Schwarz et al., in U.S. Patent No. 5,026,650, both of which are hereby incorporated by reference herein in their entirety.

The invention will now be described in greater detail with reference to the following non-limiting examples.

Example 1

This example describes the process of establishing a primary culture from highly purified adult islets and a method to induce endocrine cell selection through the use of specific defined media (VRX-E: "primary establiεhing media") in a cell culture environment which allows for adherence of endocrine cells and negative selection for fibroblasts.

In accordance with the present invention, it has been discovered that in order to accomplish endocrine cell selection of terminally differentiated, adult cells, aε well aε to induce proliferation of such cells, it is necessary to provide an environment which simulates that of the pregnant state, as well as to provide specific growth factors for beta cell selection. In addition, an environment is provided whereby such endocrine cells could attach either to a specifically coated tissue culture flask with materials such as matrogel, hyaluronic acid, laminin, and collagen. The media, which is described below, reflects the discovery that growth factors mimicking that of the pregnant state, such as human placental lactogen, growth factors from extracts of the pituitary and hypothala us, and factors specific for beta cell of selection (hepatocyte growth factor) , are critical. Establishing media (VRX-E) is the base media set forth in Table 1, containing components 1-58 thereof. Derivatives or variants of the base media are then prepared by adjusting (either by addition or subtraction or a combination thereof) the base media, for example, by adding one or more of the optional components 59-61 and/or by deletion of such additives as (13) , (56) , (57) and/or (58) .

Table 1

MANDATORY COMPONENTS:

1. Glucose 1.0000 g/L

2. Hypoxanthine 0.0047 g/L

3. Folic acid 0.0010 g/L

4. Thymidine 0.0007 g/L

5. Transferrin 5.0 μg/ml

6. Insulin 10.0 μg/ml

7. Hydrocortisone 3.5 ng/ml

8. Selenous acid 2.5 ng/ml

9. Calcium chloride 0.03885 g/L

10. Magnesium sulfate«7H₂0 0.060 g/L

11. Magnesium chloride 0.060 g/L

12. Human placental 0.001- -0.020 g/L lactogen

13. Hypothalamus extract 0.03-0.10 g/L (protein)

14. L-alanine 0.018 g/L

15. L-arginine HCl 0.422 g/L

16. L-asparagine anhydr. 0.030 g/L

17. L-aspartic acid 0.026 g/L

18. L-cystine HC1«H₂0 0.070 g/L

19. L-glutamic acid 0.030 g/L

20. L-glutamine 0.292 g/L

21. L-glycine 0.016 g/L

22. L-histidine HC1«H₂0 0.042 g/L

23. L-isoleucine 0.008 g/L

24. L-leucine 0.026 g/L

25. L-lysine HCl 0.073 g/L

26. L-methionine 0.009 g/L 27. L-phenylalanine 0.010 g/L

28. L-proline 0.070 g/L

29. L-serine 0.021 g/L

30. L-threonine 0.024 g/L

31. L-tryptophan 0.004 g/L

32. L-tyrosine di-Na salt 0.016 g/L

33. L-valine 0.023 g/L

34. L-ascorbic acid 0.045 g/L

35. Biotin 0.07 mg/L

36. D-Calcium pantothenate 0.5 mg/L

37. Choline Chloride 0.014 g/L

38. Myo-inoεitol 0.036 g/L

39. Niacinamide 0.04 mg/L

40. Pyridoxine HCl 0.06 mg/L

41. Putrescine•2HC1 0.3 mg/L

42. Riboflavin 0.04 mg/L

43. Thiamine HCl 0.29 mg/L

44. Vitamin B-12 1.4 mg/L

45. Na-pyruvate 0.220 g/L

46. Linoleic acid 0.09 mg/L

47. Lipoic acid 0.2 mg/L

48. Phenol red 1.2 mg/L

49. Sodium chloride 7.53 g/L

50. Potassium chloride 0.23 g/L

51. Disodium phosphate 0.135 g/L (anhydr)

52. Potassium phosphate 0.068 g/L (monobasic)

53. Cupric sulfate_'6H₂0 0.002 mg/L

54. Ferrouε sulfate«7H₂0 0.8 mg/L

55. Zinc sulfate«7H₂0 0.4 mg/L

56. Serum (fetal, bovine 10 - 50 ml/L or human)

57. Pituitary extract 0.01-0.05 g/L (protein)

58. Hepatocyte growth 5-15 ng/ml factor/scatter factor OPTIONAL COMPONENTS:

59. Nicotinamide 2.5-10 mM

60. Human serum albumin 1 g/L

61. Supplemental Calcium 0.078 g/L chloride

Islets were isolated from a pancreas retrieved from a 22 year old cadaver male donor. Following collagenase digestion, the islets were purified by density gradient centrifugation, and cultured overnight in standard cell culture media containing RPMI 1640 (Biowhittakes, Inc.) supplemented with 10% fetal bovine serum. Following 24 hours culture, the islets were collected, washed and then placed in cell culture vessels which allowed attachment, optionally including tissue culture flasks coated with either matrogel, la inan fibrinogen or standard petri dishes. The cells were fed every three to four days with 5-15 mM of VRX-E establishing growth media for a period of 14 days. Endocrine cells rapidly attached to the surface of the tissue culture vessel within 24-48 hours. By the end of 14 days a confluent cell monolayer is noted. Using both histochemical and molecular biology probes, it was confirmed that this population of cells was, indeed, endocrine cells and hormone producing. The probes used to confirm the presence of endocrine cells were mRNA for STF-l, mRNA for insulin, and immunohistochemical chemistry for anti-insulin, anti-somatostatin and anti-glucagon. In addition, as described below, functional assays using static glucose stimulation, and perifusion of the cellε, using glucose as a stimulus, demonstrated excellent physiological release of insulin, confirming the proliferation of insulin secreted beta cells.

Using this method of endocrine cell selection from freshly isolated islets, islet cell proliferation was confirmed in 57 different HLA typed, HIV negative, pancreatic donors between the ages of 15 to 65 yrs. Normal Chromosomal analysis of the islet cells proliferated at various stages of doublings demonstrated that, indeed, these cells were primary cell cultures capable of propagation without transformation.

Following establishment of endocrine cell selection, the endocrine cells thus selected were frozen by cryopreservation, and stored in liquid nitrogen as a master cell bank, and are used for further expansion in subcultures.

Example 2

Beta Cell Specific Differentiation Media: VRX-S.

Following endocrine cell selection from the freshly isolated adult islet population, as described in Example 1 above, ongoing differentiation of these cells, to retain the insulin secretory capacity, must be retained. A method to specifically induce differentiation and ongoing proliferation of the endocrine cells thus selected has been developed utilizing VRX-S media. Specifically it is the goal of this process to maintain the differentiation pathway in the direction of insulin secreting cells. It is presently believed that within the cell population selected in Example 1 above, there exist multipotential progenitor cells which can be directed to differentiate into specific insulin secreting beta cells. In accordance with the present invention, it has been discovered that a high dose nicotinamide during the early phase of proliferation is critical to induce beta cell differentiation. While it has previously been described that a high dose of nicotinamide is a potent inducer of endocrine differentiation in the fetal human pancreas, there has been no clear evidence of the effect of nicotinamide on adult islet cells.

In accordance with the present invention, it has been discovered that the timing and the concentration of nicotinamide application is critical to successful proliferation and differentiation of islets. Thus, it has been discovered that high doses of nicotinamide are needed during the early phases of the culture period to induce differentiation, but if such high doses are maintained during the extended proliferation phase (described below in Example 3) , nicotinamide is observed to be toxic to the cells and is, therefore, highly undesirable at this specific stage of cell growth. Thus, not only is the nicotinamide dose important, but the timing of nicotinamide application is also seen to be very important during the growth phase of induced differentiation. This observation has not been previously reported in the art, and gives rise to the beta cell specific differentiation media defined below, utilizing different concentrations of nicotinamide.

Thus, VRX-s (specific beta cell proliferation medium) is formulated by the addition of the following amounts (g/L) of nicotinamide to 1000 ml of VRX-E (to produce the concentrations shown in parenthesis below) :

VRX-S1 1.22 (10.0 mM)

VRX-S2 0.61 (5.0 mM)

VRX-S3 0.305 (2.5 mM)

Glucose stimulated insulin secretion in adult human islet cells cultured in consecutive subcultures was examined, with and without nicotinamide. After 14 days of primary culture utilizing the VXR-E establishing media described above, the cells were reseeded in VRX-S media, in the presence and absence of nicotinamide (10 mM) , and subcultured for five weeks with weekly passaging.

Triplicate cultures (0.25 M cells/56 cm petri dish) were tested. On day four of each passage, a sterile static glucose stimulation (SGS) test was performed. The sterile static glucose stimulation test was carried out as follows: First the cells were washed with glucose free Krebs Ringer bicarbonate (KRB) buffer (containing 100 mg/ml human serum albumin) , then incubated with basal 60 mM glucose (KRB 60) buffer at 37°C in a C0₂ incubator. After 60 minutes the supernatants were saved and replaced with 350 mg/ml glucose (KRB 350) buffer for the next 60 minutes. Incubation was completed with a final 60 minutes KRB 60 exposure. Supernatants were tested for insulin content by radio immunoassay (RIA) . Following the test, cells were fed with fresh medium and cultured for three more days before pasεaging. On day εeven, cellε were trypsinized, counted and reseeded with the same density (0.25 M/plate) . Insulin concentration is calculated in μIU/1.0 million cells/60 min.

RIA analysis demonstrated that the cells responded to glucose by insulin secretion as shown in Table 2.

As can be seen from Table 2, at 19- and 256-fold expansion, the use of nicotinamide is advantageous for increasing insulin secretion from the proliferated cells. Thus during this fold of expansion the use of nicotinamide at the appropriate concentrations (i.e. VRX-S beta cell specific differentiation media) is critical. However, the continued use of this media for further expansion is deleterious because of the negative and toxic affects of nicotinamide during these advanced phases. Thus for the extended proliferation media, nicotinamide is not used at these high doses. See Example 5 for further discussion of nicotinamide doses.

Example 3

This example illustrates the importance of scatter factor for the maintenance of beta cell phenotype in proliferating adult islet cell culture, especially during the phase of specific beta cell selection (i.e., employing VRX-S media) . Islet cell cultures were initiated and established in VRX-E media as described in Example 1. Cells were then subcultured in VRX-S media as described in Example 2. Control cells were submitted to subculture in VRX-S medium, but without the addition of scatter factor. Adherent cells at 50% confluency were fixed and processed for insulin with immunocytochemistry.

Immunocytochemical staining involved a two stage peroxidase procedure with peroxidase-conjugated secondary antibodies. First antibodies were Guinea Pig anti-insulin, rabbit anti-glucagon, and rabbit anti-somatostatin. Peroxidase reaction was developed with 3,3-diaminobenzidine (DAB) substrate and 0.01% hydrogen peroxide (Vector Lab). Counterstaining was performed with Gill's Hematoxyline (blue nuclei) . As can be seen from photomicrographs, beta cells which are stained positively for insulin are also seen to enter the mitotic cycle. This demonstrates clearly that adult differentiated insulin secreting cells have the capacity, under the appropriate conditions, to divide. This is the first clear demonstration that, with the use of scatter factor, beta cell specific selection is induced.

Positive insulin staining cells can be observed entering into late metaphase, telophase, and late telophase (up to completion of cytokinesis) . The effect of scatter factor is clearly seen when photomicrographs taken of cells grown in the presence or absence of scatter factor are compared. Thus, VRX-S media (i.e., nicotinamide (10 mM) and scatter factor (10 ng/ml) are added to the establishing media, which demonstrates that insulin secreting cells, as evidenced by positive light brown insulin staining, form beta cell nests which are at different stages of mitosis. In contrast, control cells (i.e., without the addition of scatter factor) , show quiescent beta cells with positive insulin staining and the absence of mitosis. As noted above, this is the first demonstration that adult fully differentiated beta cells have the capacity to undergo mitosis. While investigators in the past have shown the ability of fetal tissue to proliferate and divide, the demonstration of proliferation of adult isletε with ongoing insulin production, as evidenced by a positive insulin stain, has not previously been demonstrated.

Example 4

This example illustrates the importance of scatter factor in the preservation of beta cell function during proliferation. Thus, islets were isolated from seven different donors, cultured for 14 days in establishing medium then trypsinized and reseeded in VRX-S beta cell specific proliferation medium. Control cultures were set up without scatter factor. Sterile SGS was performed with Glucose (350 mg/dl) plus Theophylline

(lOmM) , and insulin secretion was measured by RIA. Table

3 shows the significant increases in stimulated insulin secretion in the presence of scatter factor.

Table 3

Glucose + theophylline stimulated insulin secretion of proliferating islet cells cultured with and without scatter factor

Insulin (μIU/1.0 M cells/60 min)

Cell expansion 20 fold 200 fold 1000 fold

ID# SF no SF SF no SF SF no SF

HD-183 953.0 703.0 518 285 330 127

HD-185 3990.0 3640.0 1120 845 785 492

HD-186 1255.0 480.0 398 169 287 122

HD-189 666.6 208.0 216 132 63 30

HD-192 500.0 493.0 334 273 133 119

HD-196 788.0 657.0 423 388 244 208

HD-197 321.0 200.0 165 130 108 96

MEAN 1210.5 911.6 453.4 317.4 278.6 170.6

As noted previously, this is the first demonstration of the ability of fully differentiated adult islet to undergo 20-, 200- and even 1,000-fold expansion. Furthermore, under the conditions (and employing the media described herein) ongoing insulin production and insulin secretion in response to glucose stimulation is now demonstrated. Heretofore, the ability of adult islet cells to undergo this level of expansion has never been demonstrated.

Example 5

This example illustrates the islet cell growth pattern and extended cell proliferation utilizing a media designed specifically for this purpose (VRX-P media) . Thus, islets were cultured for 14 days in VRX-E establishing medium then cells were trypsinized and reseeded in VRX-S beta cell specific proliferation medium for the next 14 days. On day 28 cells were passaged into VRX-P medium (i.e., a medium with lower concentrations of nicotinamide, and optionally without the addition of scatter factor) and grown in εerial culture for cell expansion. In accordance with the present invention, it has been discovered that once beta cell specific proliferation has been induced, extended propagation for one, two or even three months could be achieved without mutation or chromoεomal aberration employing VRX-P medium. VRX-P medium is a modification of the beta cell specific proliferation media (VRX-S medium) in that scatter factor can optionally, at this stage, be removed and the nicotinamide concentration is reduced to a range of about 0.0003 mM to 5.0 mM.

An initial inoculum of 500 islets per standard 100mm petri dish was used, with passage ratios of 1:3 per pass, which occurred approximately on every 3-4 days. On day 70 growth factors were withdrawn and cells were incubated in VRX-C for two more weeks. In accordance with the present invention, it has been demonstrated that, after an extended proliferation phase, growth cessation can be accomplished by removal of growth factors. The media employed for this purpose is referred to herein as VRX-C media, which is the VRX-E medium, absent the growth factors. Evidence of cessation of growth, as well as normal chromosomal analysis, confirmed that these ware primary cell cultures, and not transformed tumor cell lines.

The distinction between primary cell cultures and transformed tumor cell lines is important in that, to date, substantial efforts have been invested in the possibility of developing tumor cell lines in attemptε to develop an unlimited supply of insulin producing cells. In contrast, the final product of the invention process is significantly different. Thus, the proliferated cells prepared according to the invention are not tumor cell lineε, but instead are normal non-transformed cells which have undergone mitosis.

Figure 2 graphs the growth pattern of pancreatic cells up to 88 million fold expansion. The Figure also showε that cell propagation εtopped after growth factor withdrawal (i.e., converεion to VRX-C media). The growth curve was generated by cell counting after trypsinization and reεeeding 0.5M cellε per diεh. Cell viability waε checked at each pasε with trypan blue exlusion and was always >90%.

Example 6

This example illustrates that the average population doubling time of normal adult islets is about

2.7 days. Using the procedures outlined in Figure 1 and described in Examples 1-5, pancreatic cell growth was carried on for a total of 70 days. Population doubling time was calculated at each pasεage and plotted on the Y axis (See Figure 3) .

This finding of doubling in 2.7 days, with fully differentiated adult islets, is novel. As noted previously, heretofore only tumor cell lines or fetal tissue has been shown to have the ability to mytose at a rate close to that described herein. Thus, in accordance with the present invention, it has been demonstrated that by the use of appropriate growth factors, coupled with provision of a suitable environment for the cells, excellent doubling times can be achieved, while the cells retain their degree of differentiation with ongoing insulin secretion.

Example 7

This example illustrates the importance of scatter factor for the preservation of insulin message in proliferating islet cells. Reverse transcriptase polymerase chain reaction (RT PCR) was applied to follow insulin message in proliferating islet cells. Total RNA was extracted from proliferated islet cells at consecutive pasεageε after 16-, 64-, 256- and 1024-fold expanεion with TriReagent (Sigma) and equivalent amounts (0.2mg) were subjected to RT PCR analysis using primers specific for insulin and beta actin (control) . All RT PCR products were amplified in parallel and analyzed by electrophoresis on 1.5% agarose and viεualized with SYBR Green. The insulin product (420 base pairs) and the beta actin product (661 base pairs) can readily be distinguished.

RT PCR results show that insulin message is retained in media containing scatter factor, even at greater than 100-fold expanεion. In contraεt, media without scatter factor show disappearance of insulin message at that stage of expansion. Example 8

This example illustrates the successful production of islet cell clusters (pseudo isletε) by aggregation of proliferated and growth ceased adult islet cells in a three dimensional, rotational wall vessel with low shear stress.

The assembly of functional islet cell clusters from proliferated single islet cells is problematic, due to the effects of shear stress, turbulence and inadequate oxygenation in conventional spinner flask or roller bottle vessels. In accordance with the present invention, it has been discovered that a vesεel with an integrated rotating wall (RWV) and a slow turning, lateral vessel (e.g., (such as, for example, the device disclosed by Schwarz et al., in U.S. Patent No. 4,988,623, or the device disclosed by Schwarz et al., in U.S. Patent No. 5,026,650, both of which are hereby incorporated by reference herein in their entirety) solveε thiε problem. The function of both veεsels is baεed on two deεign principles: the vessel is rotating horizontally when it is filled completely with culture media and the cells are oxygenated by a silicon rubber membrane. As the vessel rotates, the liquid inside quickly accelerates and the fluid maεε rotates at the same angular rate. This environment eliminates destructive shear gradients. Cells obey simple kinematics and uniformly suspend in the fluid (Prewett et al., J. Tissue Cult . Meth . 15:29-36 (1993)).

At an advanced stage of cell culture, e.g., after 12,000-fold expansion and two weeks growth cesεation period, cells were released from T flaskε with trypεinization and resuspended in VRX-A medium. After counting and viability tests, 250 million cells were transferred to a vessel with a rotating wall. The cell density was determined by limitation of oxygenation. Sampleε obtained hourly were monitored for pH, C0₂, 0₂ and glucoεe metabolism. As the glucose metabolic rate increased (i.e., lactate >0.8g/L, glucose <0.8 g/L) nutrients were replenished by partial media changeε. Oxygen tenεion waε kept between 80-120 mmHg, carbon dioxide tenεion was kept between 30-40 mmHg, and pH maintained between 7.2 - 7.6. The three dimensional aggregation of islet cells was then monitored and determined beginning at 6 hr and continued for a 42 hr period. The number of aggregates was determined in 35 mm petri dishes in islet size categorieε (>300, 200-300, 100-200 and 50-100 μm) . The size distribution of aggregates can readily be determined by photomicrograph, which revealε aggregateε of several cells up to large aggregates approaching hundreds of cells.

Thus, means for reconfigurating islets in a proliferated state into a three dimensional structure have been demonstrated. This is accomplished employing a cell culture vesεel with a rotating wall and a vessel which imparts low shear stress to the cells aε they aggregate.

The media uεed during thiε phase of aggregation is defined aε VRX-A. To make VRX-A (aggregation medium) do not add serum, pituitary extract or scatter factor to the basic formulation, but add: Nicotinamide 0.305 g/L

Human εeru albumin 1.00 g/L

Supplemental calcium chloride 0.078 g/L

Example 9

This example describeε functional tests of islet cell aggregateε after 12,000-fold expanεion. Iεolated iεlet function iε characterized by the insulin secretion response to glucoεe challenge, where glucose stimulation can be carried out in dynamic and static circumstances. Dynamic glucose stimulation (Figure 5) was performed in a perifusion (PF) system. The syεtem consisted of two reservoirs containing KRB-60 and KRB-350 + theophylline (lOmM) , a cassette pump, tubing set with a Swinnex filter holder (13mm) and 3 way stopcock and fraction collector. The solutions and the filter holder were immersed into a 37°C waterbath. 50-100 islet equivalent pseudo isletε (150 μm)-were placed on a 20 μm pore size εterile filter in the filter holder and exposed to continuous fluid flow of KRB buffer with a flow rate of 1.0 ml/min. After a (30, 60 or 90 min) equilibration, time effluents were collected. After 30 min low glucose medium was changed to a stimulatory glucose solution. Perfusion was finished with basal glucose medium.

For εtatic glucose stimulation (SGS; see Figure

4) 50-100 islet equivalent pseudo isletε (150 mm) were placed on a 40 mm εterile filter in 6 well plates. Islets were incubated in KRB 60 solution to determine basal insulin secretion. Following incubation for 60 min at 37°C, the filters holding the islets were transferred into KRB 350 solution with 10 mM theophylline. The asεay was finished in a second KRB 60 incubation. Supernatants were collected for insulin determination.

Figure 5B shows the rate of insulin release from pseudo iεletε aggregated for 48 hourε. Figure 5A shows the normal insulin secretion rate of nonproliferated islets.

Example 10 Example of Western Blot Analysis for STF-l

In the pancreas, STF-l is only present in beta cells and delta cells. The presence of STF-l protein was used to characterize the proliferating cells. Cultures of proliferated iεlets (10 cm plates) at passage 1 and pasεage

4 were extracted at 65°C with 0.5 ml each Cell Lysis Solution (1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM 2-mercaptoethanol, 100 mM KCI, 20 mM Triε HCl pH 7.4). Equivalent amounts of each extract (10 mg protein) were electrophoresed on a 10% SDS polyacrylamide gel and transferred to nitrocellulose. The blot was probed with a STF-l specific antibody (Peers et al., Mol Endocrinol 8:1798-1806 (1994)) (1:1000 in 1% gelatin, 0.02% Tween 20, 500 mM NaCl, 20 mM Tris HCl pH 7.5) and detected with [1251] protein A (Amerεham IM 144, 1:1000 in 20 mM Tris HCl pH 7.5, 500 mM NaCl, 0.5% bovine serum albumin, 0.5% Triton X 100, 0.2% SDS) followed by autoradiography. There was no observed change in the relative levelε of STF-l protein from passage 1 to passage 4 indicating that the proliferated cells are islet cellε.

Example 11

Thiε example illuεtrateε the quantitation of STF-l mRNA levelε as probes specific for beta cells. Proliferated beta cells from representative islet cultures are extracted with TriReagent (Sigma #T 9424) and both RNA and protein samples are prepared. The mRNA levels of STF-l are determined by quantitative Reverse Transcriptase Polymerase Chain Reaction (RT PCR) utilizing primerε that diεtinguish the correctly spliced message. Northern blot analyses of selected samples can be used to confirm the RT PCR results. High level STF-l expression is seen in cultures containing predominantly pancreatic endocrine cells but not in cultures of acinar cells or fibroblaεts. By comparing the expresεion of STF-l with other markerε (e.g. inεulin and εomatostatin) the proliferative capacity of the beta cells in the culture can be determined and optimized for each experiment. The culture conditions for each donor islet population can be tailored based upon these reεultε. Example 12

This example demonstrates the use of the level and activity of STF-l protein to optimize beta cell culture medium. The interaction of STF-l with the CT2 element in the insulin promoter is augmented in insulinoma cells following exposure to glucose, implying that both the level and state (phosphorylation, glycosylation, etc.) of the STF-l protein is important for insulin expression. Islet cell culture conditions were optimized in part based on the expression level and glucose dependent modification of the STF-l protein. The levels of STF-l were determined in the protein extracts by Weεtern blot analyεeε using specific antibodies (Peers et al., Mol Endocrinol 8:1798-1806 (1994)) and compared with control proteins and with other markers. Under optimal islet cell proliferation conditions, STF-l protein levels remain constant. Glucose dependent modifications of STF-l were examined in cell extracts by electrophoretic mobility shift assays, DNA footprint experiments, and by isoelectric focusing. The results of the assays were compared with SGS and perfusion experiment data to correlate observed changes in STF-l with glucose responsive inεulin release. Culture conditions were then changed as appropriate to achieve optimal levels.

In addition, islet cells are identified in representative cultureε by in situ hybridization with a STF-l probe. Co-localization of STF-l and other beta cell markerε in cultured cells allow the morphological identification of inεulin producing cells, leading to the sub-culturing of homogeneous beta cell clones. Physical isolation of these clones may be possible by robotic instrumentation, including the use of laser directed splicing of the cell and retrieval of these cloneε from the cell culture vessel for further subculture. The use of STF-l as a probe to confirm specific beta cell εelection and to optimize media for beta cell selection has not previously been deεcribed.

Example 13

Thiε example demonstrates regulation of the STF-l promoter. It iε preεently believed that high level expreεεion of STF-l is required for expresεion of the beta cell phenotype. In a tranεgenic mouεe model, a STF-l promoter fragment haε been shown to direct the expression of a beta-galactosidase reporter gene predominantly to pancreatic beta cells. It is thus possible to introduce a similar reporter construct into proliferating beta cell populations and monitor the effect of media components in order to identify factors that maximize transcription for the STF-l promoter. Reporter gene expression data can be correlated with the other data and conditions optimized for glucose dependent insulin release.

Example 14

This example demonstrateε the increased efficacy of insulin secretion following long term culture using freshly isolated isletε co-cultured with proliferated iεlets. Thus, freshly isolated isletε, together with proliferated islets, were encapsulated in an alginate-based membrane and placed in an incubator for an extended culture period of 30 days. The performance of this combination of isletε waε compared to the performance of proliferated islets encapsulated alone.

In accordance with the present invention, it haε been diεcovered that iεlets which are co-encapsulated (i.e., freshly isolated iεletε, combined with proliferated iεlets) demonεtrate an ability to maintain insulin output for an extended period of time when so cultured. Aε can be εeen in Figure 6, when proliferated islets from donor 186 were co-encapsulated with freshly isolated islets from donor 180, excellent insulin secretion was noted at day 150 and even day 180 of culture. In contrast, when islets from donor 186 were encapsulated and cultured alone, a significant drop in insulin output was noted in day 123 (even though good insulin output was noted in response to glucose stimulation at day 89) . Thus, it has been discovered that co-culturing freshly isolated islets with proliferated islet aggregates appear to enhance the long term viability of the islets. It is presently believed that certain factors from the proliferated and freshly isolated islets may act synergistically in enhancing survival and function of these cells.

The ability to co-culture other cell types from the pancreaε with theεe iεlets (e.g., ductal and acinar cellε with proliferated islets) has also been explored. It would appear that the addition of these other cell types may have an enhancing value on the long term viability of these cells. Theoretically thiε appears reasonable since, in its natural state, an islet is surrounded by other cell types (i.e., acinar, ductal and endothelial cells). Thus, additional experiments have been performed whereby fibroblasts and endothelial cells have also been co¬ cultured with iεlets. As with prior co-culture results, these proliferated islets, as well as these combined cell types, have shown long term viability.

It is possible that endothelial cells from the recipient may one day be retrieved and co-cultured with proliferated isletε from the donor, and in thiε way provide a tolerant hybrid organ which may or may not require immunoεuppreεεion. Example 15

This example demonstrates the use of encapsulated proliferated human islets for transplant into a type 1 diabetic patient. Thus, proof of principle haε been demonεtrated by the uεe of encapεulated proliferated human islets in a type 1 diabetic patient. The first patient to receive encapsulated proliferated human islets was a 41- year-old male with insulin dependent diabetes for 33 years requiring a mean +10 era of 0.6 +0.01 units of insulin per kg per day. The patient suffered from severe complications of the disease, including a history of proliferal neuropathy, retinopathy, and end stage renal failure resulting in a living related kidney transplant.

Through a 2cm abdominal incision, encapεulated human iεletε (60% of which were derived from proliferated source) were injected into the peritoneal cavity. Thus, this patient received a dose of co-encapsulated, freshly isolated and proliferated islet cell aggregateε. The patient demonεtrated immediate islet function from the interperitoneally transplanted cells. Following injection of 12,000 isletε per kg, insulin secretion waε noted within 24 hourε. The mean εerum glucoεe levels fell from a pre transplant level of 232 +9.5 mg per dl to 116 +18.6 mg per dl on the first post operative day, with all exogenous insulin being essentially discontinued. The patient thereafter maintained a stable daily mean blood glucose level ranging from 116 +18.6 to 155 +12 mg per dl with less lability relative to hiε pre-tranεplant levels, while on a significantly (P <0.001) reduced dose of insulin of approximately 0.05 to 0.2 units per kg per day for a period of three weeks. Fasting pro insulin levels and c peptide levels were significantly increased.

This evidence of immediate islet function from proliferated human isletε iε the firεt demonstration that isletε expanded in vitro can function when tranεplanted in man. The patient, over a two month follow up period, then demonstrated a significant improvement in glycosylated albumin level from a pre transplant level of 5.4% to 3% at six weeks, confirming improvement in his glycemic control as a result of the transplant. The pro-inεulin level increased from a non- detectible range of >0.25 nanograms per ml pre transplant to 6.47 nanograms per ml on day 3, 2.75 nanograms per ml after two weeks, and 0.94 nanograms per ml at six weeks.

Example 16

This example demonstrates the proliferation of non-human adult on neonatal porcine islet cells. Islets were isolated from adult porcine pancreata, as well as from neonatal pancreata, by standard collagenase digestion techniques and purified uεing a density grade inseparation. Following purification of these islet cell masses, they were subjected to the same culture techniques aε deεcribed in Example 1, uεing the VRX-E eεtablishment media, followed by the VRX-S beta cell specific media. Using these non¬ human islet cell populations, similar evidence of proliferation and mitosis as demonstrated in examples deεcribed above uεing human iεletε was noted.

While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

Claims

That which is claimed:

1. A method for stimulating the ex vivo proliferation and differentiation of adult pancreatic islet beta cells, said method comprising:

(a) contacting a primary culture of adult pancreatic cells under conditions suitable to induce beta cell proliferation; and

(b) contacting the differentiated cells produced in step (a) under conditions suitable to induce prolonged proliferation of said cellε.

2. A method according to claim 1 further compriεing:

(c) contacting the proliferated cellε produced in εtep (b) under conditions suitable to arrest the growth thereof.

3. A method according to claim 2 wherein said conditions suitable to arrest the growth of said cells comprise culturing said differentiated/proliferated cells in growth cessation media, VRX-C.

4. A method according to claim 2 further comprising:

(d) culturing the cells produced in εtep (c) under conditions suitable to promote the formation of three dimensional tissue-like structureε.

5. A method according to claim 4 wherein εaid conditions suitable to promote the formation of three dimensional tissue-like structures comprise culturing in a microgravity device.

6. A method according to claim 5 wherein said culturing is carried out in the presence of aggregation media, VRX-A.

7. A method according to claim 1 wherein said primary culture of adult pancreatic islet cells is produced by culturing islets in primary establishing media, VRX-E.

8. A method according to claim 1 wherein said conditions suitable to induce beta cell proliferations and/or differentiation comprise culturing said primary culture of adult pancreatic islet cells in the presence of beta cell specific differentiation media, VRX-S.

9. A method according to claim 1 wherein said conditions suitable to induce prolonged proliferation of said cells comprise contacting said proliferated and/or differentiated cells with a proliferation inducing amount of a cytokine (hepatocyte growth factor) for a suitable amount of time.

10. A method according to claim 9 wherein said proliferation inducing amount of cytokine (hepatocyte growth factor) ranges from about 5 to about 50 ng/ml.

11. A method according to claim 1 wherein said cell proliferation comprises an increase in beta epithelial cell number relative to other cell types.

12. A method according to claim 1 wherein said cell proliferation and/or differentiation comprises an increase in the formation of islet-like cell clusters containing beta epithelial cells.

13. A method according to claim 1 wherein said proliferated cellε are derived from human pancreatic iεletε.

14. A method according to claim 1 wherein said proliferated cells are derived from porcine pancreatic islets.

15. A method for stimulating the ex vivo proliferation and differentiation of neonatal pancreatic islet beta cells, said method comprising:

(a) contacting a primary culture of adult pancreatic cellε under conditionε suitable to induce beta cell proliferation; and

16. A method according to claim 15 further comprising:

(c) contacting the proliferated cells produced in step (b) under conditions suitable to arrest the growth thereof.

17. A method according to claim 16 further comprising:

18. A method according to claim 15 wherein said conditions suitable to induce prolonged proliferation of said cells compriεe contacting εaid proliferated and/or differentiated cells with a proliferation inducing amount of a cytokine (hepatocyte growth factor) for a suitable amount of time.

19. A method according to claim 18 wherein said proliferation inducing amount of cytokine (hepatocyte growth factor) ranges from about 5 to about 50 ng/ml.

20. A method according to claim 15 wherein said proliferated cells are derived from human pancreatic isletε.

21. A method according to claim 15 wherein said proliferated cells are derived from porcine pancreatic isletε.

22. A method for treating a subject with type 1 diabetes mellitus, said method comprising:

(a) contacting a primary culture of adult pancreatic cells under conditions suitable to induce beta cell proliferation;

(b) contacting the differentiated cells produced in step (a) under conditions suitable to induce prolonged proliferation of said cells;

(c) contacting the proliferated cells produced in step (b) under conditions suitable to arreεt the growth thereof;

(d) culturing the cellε produced in step (c) under conditions εuitable to promote the formation of three dimenεional tiεsue-like εtructureε containing increased numbers of insulin producing islet-like cell clusters containing beta epithelial cells;

(e) encapsulating said cells or islet-like cell clusterε; and

(f) parenterally ad iniεtering an effective amount of said encapsulated cells to said subject.

23. A method according to claim 22 wherein said parenteral administration compriseε an intraperitoneal, intraportal, intrasplenic, intravenouε, renal εubcapsular or εubcutaneous route.

24. A method according to claim 22 wherein said cells or islet-like cell cluεterε are encapsulated within alginate microcapεuleε.

25. A method for treating a subject with type 1 diabetes mellitus, said method comprising:

(a) contacting a primary culture of neonatal pancreatic cells under conditions suitable to induce beta cell proliferation;

(b) contacting the differentiated cells produced in step (a) under conditionε εuitable to induce prolonged proliferation of said cells;

(c) contacting the proliferated cells produced in step (b) under conditions εuitable to arreεt the growth thereof;

(d) culturing the cellε produced in εtep (c) under conditionε εuitable to promote the formation of three dimensional tisεue-like structures containing increaεed numberε of inεulin producing iεlet-like cell cluεterε containing beta epithelial cellε;

(e) encapεulating εaid cellε or iεlet-like cell cluεters; and

26. A method according to claim 25 wherein said parenteral administration comprises an intraperitoneal, intraportal, intrasplenic, intravenous, renal subcapsular or subcutaneous route.

27. A method according to claim 25 wherein said cells or islet-like cell clusterε are encapεulated within alginate microcapεules.

28. A method to proliferate or differentiate adult pancreatic islet cellε in clinically useful quantities, said method comprising:

(a) seeding a primary culture of adult pancreatic cellε into a microgravity veεεel;

(b) contacting said cells in said microgravity vesεel with a complete growth medium εupple ented with a proliferation inducing amount of a cytokine (hepatocyte growth factor) for a time sufficient to allow differentiation, proliferation and aggregation of said cells; and thereafter

(c) harvesting the resulting islet-like cell clusters containing beta epithelial cells from said microgravity vesεel.

29. A method according to claim 28, further compriεing:

(d) encapεulating said islet-like cell clusters.

30. A method according to claim 29 wherein said islet-like cell clusters are encapsulated in alginate microcapsules.

31. A method to proliferate or differentiate neonatal pancreatic islet cells in clinically uεeful quantitieε, εaid method compriεing:

(a) seeding a primary culture of neonatal pancreatic cells into a microgravity vessel;

(c) harveεting the resulting islet-like cell clusters containing beta epithelial cells from said microgravity vesεel.

32. A method according to claim 31, further compriεing:

(d) encapεulating εaid iεlet-like cell cluεters.

33. A method according to claim 32 wherein said islet-like cell clusterε are encapεulated in alginate microcapsules.

34. A composition for transplanting functional adult pancreatic tissue into patients, said composition comprising: a pharmaceutically acceptable vehicle, containing primary cultures of adult pancreatic islet cells which have been contacted ex vivo with a differentiation and proliferation inducing amount of a cytokine (hepatocyte growth factor) sufficient to induce an increase in cell number, an increase in the formation of islet-like cell clusterε containing beta epithelial cellε, and the retained ability to produce inεulin in reεponse to stimulus.

35. A composition according to claim 34 further comprising freshly isolated cells εelected from islets, fibroblastε, endothelin cells, acinar cells and neural cells.

36. A composition according to claim 35 wherein said freshly isolated cells are retrieved from the patient to receive said tranεplant.

37. A composition according to claim 34 wherein said pharmaceutically acceptable vehicle is an alginate microcapsule.

38. A compoεition for transplanting functional neonatal pancreatic tisεue into patientε, εaid compoεition compriεing: a pharmaceutically acceptable vehicle, containing primary cultures of neonatal pancreatic islet cells which have been contacted ex vivo with a differentiation and proliferation inducing amount of a cytokine (hepatocyte growth factor) sufficient to induce an increase in cell number, an increase in the formation of islet-like cell clusters containing beta epithelial cellε, and the retained ability to produce insulin in response to stimulus.

39. A composition according to claim 38 further comprising freshly isolated cells selected from islets, fibroblasts, endothelin cells, acinar cells and neural cellε.

40. A compoεition according to claim 39 wherein said freshly isolated cells are retrieved from the patient to receive said tranεplant.

41. A composition according to claim 38 wherein said pharmaceutically acceptable vehicle is an alginate microcapεule.

42. A media composition comprising components 1- 58 as set forth in Table 1 (VRX-E) .

43. A composition according to claim 42 further comprising 2.5-10 mM nicotinamide (VRX-S).

44. A composition according to claim 43 not including components (13) , (57) or (58) as set forth in Table 1 (VRX-C) .

45. A composition according to claim 43 further comprising about 1 g/L of human serum albumin and an added 0.078 g/L of calcium chloride (VRX-A), but not including components (13) , (56) , (57) or (58) as set forth in Table 1.

46. A composition comprising a combination of freshly isolated islet cellε and proliferated adult iεletε.

47. A compoεition according to claim 46 wherein εaid combination iε encapεulated.

48. A method for treating a subject with type 1 diabetes mellitus, said method comprising parenterally administering an effective amount of the encapsulated cells of claim 47 to said subject.

49. A method to identify proliferated and/or differentiated cells as beta cells, said method comprising monitoring said cells for the expression of STF-l.

50. A method to optimize culture media for the differentiation of epithelial cells for selection for insulin-secreting beta cells, said method comprising monitoring a cell population for the presence of STF-l as a function of variations in the culture media, and modifying said media so as to maximize expression of STF-l.

51. A method for the preparation of cell clusters from proliferated, differentiated, growth arrested cellε, said method comprising subjecting said cells to aggregation conditions in a microgravity vessel.

52. A method for the preparation of a primary culture of adult pancreatic islet cells, said method comprising culturing isletε in primary eεtabliεhing media.

53. A method according to claim 52 further comprising contacting said primary culture of adult pancreatic cells under conditions suitable to induce beta cell proliferation.

54. A method according to claim 53 further comprising contacting said differentiated cells under conditions suitable to induce prolonged proliferation thereof.

55. A method according to claim 54 further comprising contacting said proliferated cells under conditions suitable to arrest the growth thereof.

56. A method according to claim 55 further comprising culturing the growth arrested cells in a microgravity vessel under conditions suitable to promote the formation of three dimensional tissue-like structures.

57. A method according to claim 56 further comprising encapsulating said three dimensional tissue-like structures.

58. A method for the preparation of a primary culture of neonatal pancreatic islet cells, said method comprising culturing iεletε in primary eεtablishing media.

59. A method according to claim 58 further comprising contacting said primary culture of neonatal pancreatic cells under conditions suitable to induce beta cell proliferation.

60. A method according to claim 59 further comprising contacting said differentiated cells under conditions suitable to induce prolonged proliferation thereof.

61. A method according to claim 60 further comprising contacting said proliferated cells under conditions suitable to arreεt the growth thereof.

62. A method according to claim 61 further compriεing culturing the growth arrested cells in a microgravity vessel under conditions suitable to promote the formation of three dimenεional tiεsue-like structureε.

63. A method according to claim 62 further comprising encapsulating said three dimensional tissue-like structureε.

64. The cellε produced by the method of claim 1.

65. The cellε produced by the method of claim 4.

66. The cells produced by the method of claim 9.

67. The cells produced by the method of claim

13.

68. The cells produced by the method of claim

15.

69. The cells produced by the method of claim

17.

70. The cells produced by the method of claim

18.

71. The cells produced by the method of claim

20.

72. The cells produced by the method of claim

28.

73. The cells produced by the method of claim

30.

74. The cells produced by the method of claim

31.

75. The cells produced by the method of claim

33.

76. The cellε produced by the method of claim 51.

77. The cellε produced by the method of claim 52.

78. The cellε produced by the method of claim 58.