CA2203032A1 - Isolated porcine pancreatic cells for use in treatment of diseases characterized by insufficient insulin activity - Google Patents

Abstract

Isolated porcine pancreatic cells, isolated populations of such cells and methods for isolating and using the cells to treat subjects with diseases characterized by insufficient insulin activity are described. The porcine pancreatic cells are preferably non-insulin-secreting porcine pancreatic cell having the ability to differentiate into aninsulin-secreting cell upon introduction into a xenogeneic subject, such as a human subject. Such cells include embryonic porcine pancreatic cells obtained from embryonic pigs between about day 31 and day 35 of gestation. The porcine pancreatic cells can be modified to be suitable for transplantation into a xenogeneic subject, for example, by altering an antigen (e.g., an MHC class I antigen) on the cell surface which is capable of stimulating an immune response against the cell in the subject (e.g., by contact with an anti-MHC class I antibody, or a fragment or derivative thereof). The isolated porcine pancreatic cells of the invention can be used to treat diseases characterized by insufficient insulin activity, e.g., Type I and Type II diabetes, by administering the cells to a subject having such a disease.

Description

CA 02203032 l997-04-l7 WO 96/12794 PCT/US9~5/12877 ISOLATED PORCINE PANCREATIC CELLS FOR USE IN TREATMENT OF
DISEASES CHARACTERIZED ~Y INSUFFICIENT INSULIN ACTIVITY

Back~round of the Invention Idlopathic or primary diabetes mellitus is a chronic disorder of carbohydrate, fat, and protein metabolism characterized in its fully expressed forrn by an absolute or relative insulin deficiency, fasting hyperglycemia, glycosuria, and a striking tendency toward development of atherosclerosis, microangiopathy, nephlo~ y, and n~ulo~lly. Underutilization of glucose is characteristic of all diabetic patients, but only some have a clearly defined severe insulin deficiency resulting from a loss of ~ cells. The large remainder of diabetic patients suffer from some impairment of insulin secretory response associated with a marked resistance to insulin in the peripheral tissues.
The phrase "idiopathic diabetes mellitus" embraces a heterogeneous group of disorders having in common the above-described characteristics. At least two major as well as several less common variants of the disease have been identified. One major variant, insulin-dependent diabetes mellitus (IDDM) (Type I). accounts for about 10% of diabetics.
A second major variant, non-insulin-dependent diabetes mellitus (NIDDM) (Type II) represents the rem~ining 90% of all diabetic patients. Robbins, S.L. et al. Pathologic Basis of Disease, 3rd Edition (W.B. Saunders Company, Philadelphia,1984) p. 97 Absent regular insulin replacement therapy using exogenously produced insulin and/or careful monitoring of the diet of diabetic patients. such patients experience a wide range of debilitating symptoms, some of which can progress into coma and ultimately death.
An alternative method of treating diabetes presently under investigation. which does not require repeated ~lmini.~tration of insulin and/or strict monitoring of diet. is transplantation of pancreatic cells or tissue from a donor to the diabetic patient. A major problem in pancreatic cell or tissue transplantation from one human to another for treatment of diabetes, however, is a shortage of donor tissue. Thompson. S.C. et al. (1990) Transplantation 49(3):571-581. Moreover, human pancreas will inevitably remain in limited supply and be subject to many constraints, including, especially with human fetal pancreatic cells or tissue. sensitive ethical issues. The improvement in patients and graft survival following human pancreas transplants (Sutherland, D.E.R. et al. (1987) Transplant. Proc.
19:113) will aiso mean that adult cadaveric pancreas may be more difficult to obtain for experimental purposes.
As a result of the above-described problems associated with transplantation of pancreatic tissue from a human donor to a human recipient. alternative sources of pancreatic tissue for transplantation have been investigated. Several groups of investigators have conducted research involving the use of pancreatic cells and tissue from animal sources. such as swine. for transplantation. See e.g, Korsgren. O. et al. (1993) Surgery 113:205-214;
Braesch. M.K. et al. (1992) Transplant. Proc. 24(2):679-680: Grotll. C.G. et al. (1992) CA 02203032 l997-04-l7 WO 96/1279`1 PCT/US9~/12877 Transplant. Proc. 24(3~:972-973; Liu, X. et al. (1991) Diabetes 40:858-866; Korsgren, O. et al. (1991) Diabetologia 34:379-386, Yoneda, K. et al. (1989) Diabetes 38 (Supp. 1):213-216; Wilson, J.D. et al. (1989) Diabetes 38 (Suppl. 1):217-219; Korsgren, O. et al. (1989) Diabetes 38 (Suppl. 1):209-212; Korsgren, O. et al. (1988) Transplantation 45(3):509-514;
Sasaki, N. et al. (1984) Transplantation 38(4):335-340. Several ofthese investigators report transplantation of pancreatic tissue samples cont~ining insulin-secreting and at least partially dirrel~"li~te~l porcine pancreatic cells into xenogeneic subjects after short-term culture.
However, these short-terrn cultures of pancreatic cells often display eventual necrosis (Thompson, S.C. et al. (1990) Transplantation 49(3):571-581)? developmental stagnation (Liu, X. et al. (1991) Diabetes 40:858-866; Korsgren, O. et al. (1989) Diabetes 38 (Suppl.
1):209-212; Korsgren, O. et al. (1988) Transplantation 45(3):509-514), decreasedproliferation (Liu, X. et al. (1991) Diabetes 40:858-86G; Korsgren, O. et al. (1988) Transplantation 45(3):509-514)~ and decreased insulin production (Yoneda, K. et al. (1989) Diabetes 38 (Supp. 1):213-216: Korsgren, O. et al. (1988) Transplantation 45(3):509-514).
Summarv of the Invention The present invention provides porcine pancreatic cell(s) which can be used to generate populations of cells useful for transplantation into diabetic subjects. The porcine pancreatic cells of the invention are capable of proliferating in vitro and in vivo and are insulin-secreting after transplantation into a recipient subject. Accordingly~ the invention pertains to isolated non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject. In one embodiment, the non-insulin-secreting porcine pancreatic cells are embryonic pancreatic cells isolated during certain stages of gestational development. It has been discovered that such porcine embryonic pancreatic cells can be m~int~ined in culture if sub-confluent and will proliferate for long periods of time, e.g., six months or more, without forming pseudo islet-like aggregates. Preferably, the pancreatic cells are obtained from embryonic pi~s at an early stage of development ~i.e., prior to forrnation of islets in vivo) and are m~int~ined in culture to allow cell proliferation without substantial differentiation into islet-like aggregates.
Moreover, the culture is not diluled out by non-insulin-producing cells, e.g.~ endothelial cells~
which are involved in islet formation. Furthermore, proliferation of the cultured pancreatic cells can be substantially augmented by adding certain embryonic proliferating agents to the culture. These embryonic proliferating agents can decrease the doubling time of the cells by almost two-fold. When the cells are allowed to reach confluence~ they begin to form pseudo islet-like aggregates which produce insulin, glucagon~ and somatostatin.
Thus. large populations of non-insulin-secreting porcine pancreatic cells capable of proliferating and differenti~ting to produce insulin-secreting cells upon introduction into a subject can be prepared in an economical and time-efficient manner. The porcine pancreatic cells of the invention can~ therefore. serve as a convenient and plentiful source of cells for WO 96/1279~ PCT/US95/12877 ~lminictration to subjects having diseases caused by insufficient activity of a pancreatic horrnone, e.g, insulin, e.g., Type I or Type II diabetes, or enzyme.
Accordingly, the instant invention pertains to an isolated porcine pancreatic cell and a population of porcine pancreatic cells suitable for ~1minictration to a xenogeneic subject, 5 particularly a human subject. The isolated porcine pancreatic cell, alone or in a population, produces glucagon and somatostatin in certain embodiments, but does not secrete insulin.
Upon introduction into a xenogeneic subject, however, the porcine pancreatic cell proliferates and differentiates to form a population of insulin-secreting cells. Preferred porcine pancreatic cells are embryonic porcine pancreatic cells obtained from an embryonic pig at a selected 10 gestational age. The ~l~f~,.ed gestational age of embryonic swine from which to obtain pancreatic cells suitable for transplantation into xenogeneic subjects, particularly hl]m~n.c, was determined to be between about twenty (28) and about forty (40) days, more preferably between about thirty (30) and thirty-five (35) days~ most preferably between about thirty-one (31 ) and about thirty-five (35) days of gestation. It is preferred that the porcine pancreatic 15 cells be obtained from a pig which is essentially free from org~nicmc or substances which are capable of transmitting infection or disease to a xenogeneic recipient of the cells as described herein. Typically, the porcine pancreatic cells are isolated from a pig which is essentially free from at least one organism selected from the group consisting of parasites, bacteria, mycoplasma, and viruses. In addition, the porcine pancreatic cells can be modified as 20 described herein. The porcine pancreatic cells of the invention can be grown as a cell culture in a medium suitable to support the growth of the cells. In addition the porcine pancreatic cells can be inserted into a delivery device, e.g., a syringe, which facilitates the introduction of the cells into a subject.
The invention further pertains to a porcine pancreatic cell and an isolated population 25 of such cells which, in unrnodified form, have at least one antigen on the cell surface which is capable of stimulating an immune response against the cell(s) in a xenogeneic subject, for example. a human. The antigen on the surface of the porcine pancreatic cell(s) is altered to inhibit rejection of the cell(s) when introduced into a xeno=geneic subject. In one embodiment, the celi surface antigen which is altered is an MHC class I antigen. This MHC
30 class I antigen can be contacted, prior to transplantation into a xenogeneic subject with at least one MHC class I antibody, or a fragment or derivative thereof, which binds to the MHC
class I antigen on the cell surface but does not activate complement or induce lysis of the cell.
One example of an MHC ciass I antibody is an MHC class I F(ab'), fragment~ such as an MHC class I F(ab')~ fragment of a monoclonal antibody PT85. In one embodiment. the 35 porcine pancreatic cells are obtained from embryonic pigs of the preferred gestational ages described herein. Porcine pancreatic cells to be modified in this manner can be obtained from a pig which is essentially free from org;~ni.cmc or substances which are capable of transmitting infection or disease to a xeno~eneic recipient of the cells as described herein.

A further aspect of the invention pertains to methods of promoting or inducing proliferation of embryonic porcine pancreatic cells in which the cells are contacted with at least one embryonic proliferating agent which promotes or induces proliferation of the cells in vitro and/or in vivo. Preferred embryonic proliferating agents for promoting or inducing proliferation of embryonic porcine pancreatic cells include platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). Other embryonic proliferating agents include growth factors for which the embryonic porcine pancreatic cells of a certain gestational age (e.g., between about 31 and 35 days of gestation) express a receptor.
The invention also provides methods of isolating and promoting proliferation of porcine pancreatic cells in vitro prior to the ~lmini~tration of the cells to a xenogeneic subject. These methods typically include isolating porcine pancreatic cells from an embryonic pig and contacting the cells with an embryonic proliferating agent, such as PDGF, EGF or growth factors for which the embryonic porcine pancreatic cells express a receptor, which promotes proliferation of the cells. The cells are preferably isolated from an embryonic pig from about day 31 to about day 35 of gestation. In one embodiment the porcine pancreatic cells are non-insulin-secreting cells which have the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject. The cells can be a~lmini.~tered to the xenogeneic subject prior to or after in vitro formation of insulin-secreting islet-like aggregates.
The invention still further provides methods of treating diseases characterized by insufficient insulin activity, e.g. Type I and Type II diabetes~ in a subject, e.g., a human, having such a disease. In one embodiment. a subject having the disease is ~lmini.ctered an amount of a population of non-insulin-secreting porcine pancreatic cells which are obtained from an embryonic pig, e.g.. cells from an embryonic pig between about day 31 and day 35 2~ of gestation, having the ability to differentiate into insulin-secreting cells following ~imini.~tration to the subject. These cells can be modified as described herein prior to introduction into the subject. In another embodiment. a subject having a diseasecharacterized by insufficient insulin activity is ~lmini.~tered a population of modified porcine pancreatic cells of the invention or a population of porcine pancreatic cells obtained from 30 pigs which are essentially free from org~ni~m.~ and substances which are capable of transmitting infection or disease to the subject. These methods can further include the step of ~tlmini.~tering an immunosuppressive agent to the subject.

3~ Brief Description of the Drawings Figures lA-l B depicl insulin staining of fetal pig pancreatic cells in culture. Figure 1 A is a phase micrograph of fetal pig pancreatic cells in monolayer prior to formation of islet-like clusters. No insulin staining can be detected in these cells at this time. Figure lB is a phase micrograph of fetal pig pancreatic cells that were allowed to form islet-like clusters (indicated by arrows) and stained positive for insulin.
Figures 2A-2B depict grafts obtained from fetal pig pancreatic cells transplanted into nude mice. Figure 2A is a section from transplanted kidney showing the graft stained with 5 aldehyde-fuchsin. Insulin-cont~ining cells stain darkly and can be seen scattered throughout the graft. Figure 2B is a section from the same graft stained with antibodies which recognize insulin. Again, the insulin-cont~ining cells within the graft stain more darkly than the other cells. In both figures, the insulin-positive cells are indicated by arrows, the donor graft is marked (G), and the recipient mouse kidney is marked (K).
Detailed Description of the Invcntion 1. IS(:)LATED ~ELLS AND ~EL~ POPULATIONS OF THE INVENTION

15 A. Non-Insulin-Secreting Porcine Puncreatic Cells Suitable for Administration to Xeno~eneic Subjects This invention features an isolated non-insulin-secreting porcine pancreatic cell having the ability to differentiate into an insulin-secreting cell upon introduction into a xenogeneic subject. These cells can be used to treat diseases, such as Type I and Type II
20 diabetes, which are characterized by insufficient activity of the hormones, e.g., insulin, and enzymes produced by the pancreas. As used herein, the term "isolated" refers to a cell which has been separated from its natural environment. This term includes gross physical separation from its natural environment, e.g.~ removal from the donor animal, e.g., a pig~ and alteration of the cell's relationship with the neighboring cells with which it is in direct contact 25 by, for example, dissociation. Isolation does not refer to a cell which is in a tissue section. is cultured as part of a tissue section~ or is transplanted in the form of a tissue section. When used to refer to a population of porcine pancreatic cells~ the term "isolated" includes populations of cells which result from proliferation of the isolated cells of the invention.
When isolated from a donor swine~ the pancreatic cells of the invention are non-30 insulin-secreting. The phrase "non-insulin-secreting" refers to cells which do not deposit detectable or therapeutically significant amounts of insulin into their surroundings, e.g.~ tissue fluid or culture medium. Therapeutically significant amounts of insulin include amounts which are capable of reducing or alleviating at least one adverse effect or sym~tom of a disease characterized by insufficient insulin activity when an ~ro~l iate number of the 35 insulin-secreting cells are introduced in a subiect having such a disease. However~ the isolated cells have the ability to differentiate into insulin-secreting cells in vitro or upon introduction into a subject. In one embodiment. the non-insulin-secreting porcine pancreatic cells are further characterized bv the ability tO produce detectable amounts of gluca~on and/or somatostatin upon isolalion fronl the donor swine. The pancreatic cells of the invention.

when a~lmini~tered to a xenogeneic subject? "proliferate", a term which is used herein to mean reproduce or multiply, to produce or form a population i.e., a group of two or more cells.
Differentiation, as used herein~ refers to cells which have acquired functions different from and/or in addition to those that the cells originally possessed. For example, an non-insulin 5 porcine pancreatic cell can dirrerellliate, under specific conditions, into a cell which secretes insulin. As used herein, the phrase "secrete insulin" refers to cells which deposit insulin into their surroundings, e.g., tissue fluid, e.g, blood, or culture medium. A common method for analyzing tissue fluid or culture media for insulin-secretion is by radioimmunoassay. See Heding, L.G. (1972) Diabetolo~ia 8:260.
The term "subject" is intended to include m~mm~ 7 particularly hllm~n~ susceptible to diseases characterized by insufficient insulin activity. The term "subject" also includes m~mm~l~ in which an immune response is elicited against allogeneic or xenogeneic cells.
Examples of subjects include primates (e.g., hum~n.~ and monkeys). A "xenogeneic subject"
(also referred to herein as "recipient subject" or "recipient") as used herein is a subject into 15 which cells of another species are introduced or are to be introduced.
The pancreas is a mixed exocrine and endocrine gland. The exocrine portion is composed of several serous cells surrounding a lumen. These cells synthesize and secrete digestive enzymes such as trypsinogen, chymotrypsinogen, carboxypeptidase ribonuclease, deoxyribonuclease, triacylglycerol iipase, phospholipase A~, elastase. and amylase. The 20 endocrine portion of the pancreas is composed of the islets of Langerhans. The islets of Langerhans appear as rounded clusters of cells embedded within the exocrine pancreas. Four different types of cells- a, ~, ~. and ~-have been identified in the islets. The a cells constitute about 20% of the cells found in pancreatic islets and produce the hormone glucagon.
Glucagon acts on severaL tissues to make energy available in the intervals between feeding.
25 In the liver, glucagon causes breakdown of glycogen and promotes gluconeogenesis from amino acid precursors. The ~ cells produce somatostatin which acts in the pancreas to inhibit glucagon release and to decrease pancreatic exocrine secretion. The hormone pancreatic polypeptide is produced in the ~ cells. This hormone inhibits pancreatic exocrine secretion of bicarbonate and enzymes, causes relaxation of the gallbladder, and decreases bile secretion.
30 The most abundant cell in the islets, constituting 60-80% of the cells. is the ~ cell. which produces insulin. Insulin is known to cause the storage of excess nutrients arising during and shortly after feeding. The major target organs for insulin are the liver. muscle. and fat-organs specialized for storage of energy.
The language "pancreatic cell" refers to a cell which can produce a hormone or 35 enzyme normally produced by a pancreatic cell, e.g., an at least partially differentiated a. ~.
~ or ~ cell, and a cell, e.g., a pancreatic precursor cell, which can develop into a cell which can produce a hormone or enzyme normally produced by a pancreatic cell. In one embodiment~ the porcine pancreatic cells are characterized by the abilitv to produce glucagon andior somatostatin upon isolation from a donor swine. The pancreatic cells of the invention can also be cultured prior to ~lministration to a subject under conditions which promote cell proliferation and differentiation. These conditions include culturing the cells to allow proliferation and confluence i71 ~itro at which time the cells form pseudo islet-like aggregates or clusters and secrete insulin, glucagon, and somatostatin.
P~ncreatic cells of the invention are obtained from the pancreas of a donor swine (also referred to herein as "pig") such as, for example, a swine which is essentially pathogen-free as described herein. In a preferred embodiment, the pancreatic cells are obtained from the primordial pancreas (also referred to herein as "fetal pancreas" and "embryonic pancreas") of an embryonic donor swine and preferably at a selected gestational age. The selected gestational ages (the total gestation time for pig is approximately 115 days) for obtaining primordial pancreatic cells are determined based on the following criteria: the ability of the embryonic porcine pancreas structure to be identified; the viability of the cells upon isolation from the donor pig! the ability of the cells to proliferate in culture; the ability of the cells to remain undifferentiated (i.e.~ non-insulin secreting) in culture; and the ability of the cells to differentiate (i.e., secrete pancreatic hormones, e.g., insulin, and enzymes) upon introduction into a recipient subject. The preferred gestational age of embryonic swine from which to obtain pancreatic cells suitable for introduction into xenogeneic subjects, particularly humans, was determined to be between about twenty (28) and about forty (40) days, more preferably about thirty (30) and about thirty-five (35) days, most preferably about thirty-one (31) and about thirty-five (35) days of gestation. Earlier than about days 28-30 of gestation, the primordial pancreas in embryonic swine is not as easy to identify. Later than about days 35-36 of gestation. the pancreatic cells are not as easy to dissociate and are marginally proliferative to nonproliferative in culture. Thus, the preferred range for isolation of porcine pancreatic cells was determined to be between about thirty-one (31) and about thirty-five (35) days of development. This corresponds to fetal crown-to-rump (CRL) length of between 25 and 45 mm.
Pancreatic cells within the preferred embryonic age range have some or all of the following characteristics: the cells form a monolayer of adherent cells (i.e., they adhere to culture substrate, e.g., culture dish, forming fibroblast-like cells) when subconfluent; the cells (as a subconfluent monolayer of cells) are uniforrn in morphology, e.g., there are few if any cont~min~ting cells (e.g., cells that are associated with duct formation or cells that do not secrete or do not develop into cells that secrete pancreatic hormones or enzymes) and stain positive for glucagon and somatostatin but not for insulin; the cells are capable of proliferating for an extended period of time under apl)-ul~liate conditions, e.g., several months (six or more). in a growth medium and the cells are m~int~ined subconfluent; when the cells are allowed to reach confluence. they begin to form pseudo islet-like cell aggregates spontaneously and stain positive for insulin! glucagon. and somatostatin. Prior to the forrnation of isiet-like aggregales. there is no detectable insulin staining. See Figure 1 A. The ~orrnation of islet-like aggregates is necessary fûr the expressiûn of insulin. See Fi~ure IB.

WO 96tl279 1 PCT/US95/12877 Accordingly, this invention also features a population or a group of two or more cells, of non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject. The cells of the population are typically obtained from a selected area of the developing gut, e.g., the 5 primordial pancreas, which is typically identified as an unlobulated solid tissue located around the duodenal loop just below the stomach.
The cells of the invention can be grown as a cell culture, i.e., as a population of cells which grow in vitro, in a medium suitable to support the growth of the cells. The characteristics of the cells when grown as cell cultures are described herein in detail. Media 10 which can be used to support the growth of porcine pancreatic cells include m~mm~ n cell culture media, such as those produced by Gibco BRL (Gaithersburg, MD). See 1994 Gibco BRL Catalogue & Reference Guide. The medium can be serum-free but is preferably supplemented with animal serum such as fetal calf serum. A preferred medium is RPMI-1640 supplemented with fetal calf serum. The medium can be further supplemented with the 15 embryonic proliferating agents described herein to induce or promote proliferation of the porcine pancreatic cells.
As common methods of a~lmini~tering pancreatic cells to subjects, particularly human subjects, which are described in detail herein~ include injection or implantation of the cells into target sites in the subjects~ the cells of the invention can be inserted into a delivery 20 device which facilitates introduction by, injection or implantation, of the cells into the subjects. Such delivery devices include tubes. e.g.~ catheters. for injecting cells and fluids into the body of a recipient subject. In a pleft~ d embodiment, the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location. The porcine pancreatic cells of the invention can be inserted 25 into such a delivery device, e.g., a syringe, in different forms. For example, the cells can be suspended in a solution or embedded in a support matrix when contained in such a delivery device. As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent in which the cells of the invention remain viable. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion 30 media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists. Preferably, the solution is stable under the conditions of manufacture and storage and preserved against the Cont~min~ling action of microorg~ni~m~ such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol. ascorbic acid, thimerosal, and the like. Solutions 35 of the invention can be prepared by incorporating porcine pancreatic cells as described herein in a ph~rm~ceutically acceptahle carrier or diluent and. as required. other ingredients enumerated above, followed by filtered sterilization.
Support matrices in which the porcine pancreatic celis can be incorporated or embedded include matrices which are recipient-compatible and which degrade into products CA 02203032 l997-04-l7 WO 96/1279~ PCT/US95/12877 _ 9 _ which are not harmful to the recipient. Natural and/or synthetic biodegradable matrices are examples of such matrices. Nalural biodegradable malrices inciude plasma clots, e.g derived from a m~mm~l~ and collagen matrices. Synthetic biodegradable matrices include synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid. Other examples of synthetic polymers and methods of incorporating or embedding cells into these matrices are known in the art. See e.g, U.S. Patent No. 4,298,002 and U.S. Patent No.
5,308,701. These matrices provide support and protection for the fragile pancreatic cells in vivo and are, therefore, the preferred form in which the pancreatic cells are introduced into the recipient subjects.
B. Modified Porcine Pancreatic Cells and Isolated Populations of Modified Porcine Pancreatic Cells A further aspect of the invention is a porcine pancreatic cell which, in unmodified form, has at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in a xenogeneic subject. To inhibit rejection of the cell when introduced into the xenogeneic subject, the antigen on the cell surface is altered prior to transplantation. In an unaltered state, the antigen on the cell surface stimulates an immune response against the cell when the cell is ~lmini.~tered to a subject. By altering the antigen, the normal immunological recognition of the porcine pancreatic cell by the immune system cells of the recipient is disrupted and additionally, "abnormal" immunological recognition of this altered form of the antigen can lead to porcine pancreatic cell-specific long term unresponsiveness in the recipient. It is likely that alteration of an antigen on the porcine pancreatic cell prior to introducing the cell into a subject interferes with the initial phase of recognition of the porcine pancreatic cell by the cells of the host's immune system subsequent to :~lmini.~tration of the cell. Furthermore, alteration of the antigen can induce immunological nonresponsiveness or tolerance. thereby preventing the induction of the effector phases of an immune response (e.g., cytotoxic T cell generation, antibody production etc.) which are ultimately responsible for rejection of foreign cells in a normal immune response. As used herein, the term "altered" encompasses changes that are made to at least one porcine pancreatic cell antigen(s) which reduce the immunogenicity of the antigen to thereby interfere with immunological recognition of the antigen(s) by the recipient's immune system. An example of an alteration of a porcine pancreatic cell antigen is binding of a second molecule to the antigen. The second moiecule can decrease or prevent recognition of the antigen as a foreign antigen by the recipient subject's immune system.
Antigens to be altered according to the current invention include antigens on a porcine pancreatic cell which can interact with an immune cell in a xenogeneic (or allogeneic) recipient subject and thereby stimulate a specific immune response against the porcine pancreatic cell in the recipient. The interaction between the antigen and the immune cell can be an indirect interaction (e.g.. medialed by soluble factors which induce a response in the wo 96/1279~ PCT/USg5/12877 immune cell, e.g., humoral mediated? or, preferably, is a direct interaction between the antigen and a molecule present on the surface of the immune cell (i.e., cell-cell mediated).
As used herein, the term "immune cell" is intended to include T Iymphocytes, B
lymphocytes, monocytes and other antigen presçntin~; cells. In a pl~ft;l.ed embodiment, the S antigen is one which interacts with a T Iymphocyte in the recipient (e.g., the antigen norrnally binds to a receptor on the surface of a T lymphocyte).
In one embodiment, the antigen on the porcine pancreatic cell to be altered is an MHC
class I antigen. Alternatively~ an adhesion molecule on the cell surface, such as ICAM-l, can be altered. An antigen which stimulates a cellular immune response against the cell, such 10 as an MHC class I antigen. can be altered prior to transplantation by contacting the cell with a molecule which binds to the antigen. A preferred molecule for binding to the antigen is an antibody, or fragment thereof (e.g., an MHC class I antibody, or fragment thereoi). A
preferred antibody fragment is an F(ab')2 fragment. Polyclonal or, more preferably, monoclonal antibodies can be used. Other molecules which can be used to alter an antigen 15 (e.g., an MHC class I antigen) include peptides and small organic molecules which bind IO
the antigen. Furtherrnore~ two or more differem epitopes on the same or different antigens on the cell surface can be altered. A particularly preferred monoclonal antibody for alteration of MHC class I antigens on porcine pancreatic cells is PT85 (commercially available from Veterinary Medicine Research Development, Pullman WA). PT85 can be used alone to alter 20 MHC class I antigens or, if each antibody is specific for a different epitope, PT85 can be used in combination with another antibody known to bind MHC class I antigens to alter the antigens on the cell surface. Suitable methods for altering a surface antigen on a cell for transplantation are described in greater detail in F~nctm~n and Coe (1991) Science 252:1700-1702 and PCT publication WO 92/04033. Methods for altering multiple epitopes on a 25 surface antigen on a cell for transplantation are described in greater detail in U.S. Patent Application Serial No. 081220,741, filed March 31. 1994, the contents of which are incorporated herein by reference. The altered (also referred to herein as "modified") porcine cells can comprise an isolated population of cells. The characteristics of such populations are described above. The pancreatic cells to be modified can be obtained from donor swine at the 30 gestational ages described herein. Preferably, the modifications described herein are performed on porcine pancreatic cells prior to formation of islet-like aggregates or clusters.
Preferred donor swine are those which are essentiall~ pathogen-free as described herein.

C. Porcine Pa7lcreatic Cells and Isolated Populations of Porcine Pancreatic Cells 35 Obtainedfrom Essentiall~,~ Pathogen-Free Swine The invention also features a porcine pancreatic cell obtained from a swine which is essentially free from org~ni.cm~ or substances which are capable of transmitting infection or disease IO a xenogeneic recipiem. e.g.. a human recipienl. of the cells. ~ypically. porcine pancreatic cells are oblained from a swine which is essentially free from pathogens ~,vhich Wo 96/12794 PCT/USg~/12877 affect humans. For example, the pathogens from which the swine are free include, but are not limited to, one or more of pathogens from the following categories of pathogens: parasites.
bacteria, mycoplasma, and viruses. The swine can be free from, for example, parasites such as toxoplasma and eperytherozoon, or mycoplasma, such as M. hyopneumonia. Examples of 5 bacteria from which the swine can be free include brucella, listeria, mycobacterium TB, leptospirillum, and haemophilus suis. Additionally, the swine can be free from viruses such as zoonotic viruses (viruses which can be transferred from pigs to man under natural conditions), viruses that can cross the placenta in pregnant sows, and n~ulollo~)hic viruses.
Zoonotic viruses include, for example, a virus in the rabies virus group. a herpes-like virus 10 which causes pseudorabies, encephalomyocarditus virus. swine influenza Type A?
transmissible gastroenteritus virus, parainfluenza virus 3 and vesicular stomatitis virus.
Viruses that can cross the placenta include, for example, viruses that cause porcine respiratory reproductive syndrome, a virus in the rabies virus group, a herpes-like virus which causes pseudorabies, parvovirus. a virus that causes swine vesicular disease, techen (porcine 15 polio virus)~ hemm~gll~tin~ting encephalomyocarditus, cytomegalovirus, suipoxvirus. and swine influenza type A. Neurotrophic viruses include, for example, a virus in the rabies virus group. a herpes-like virus which causes pseudorabies. parvovirus, encephalomyocarditus virus, a virus which causes swine vesicular disease, porcine poliovirus (techen), a virus which causes hemmagl~ltin~tin~ encephalomyocarditus. adenovirus~ parainfluenza 3 virus. Specific 20 examples of viruses from which the swine are free include: a virus which causes (or results in) porcine respiratory reproductive syndrome, a virus in the rabies virus group. a herpes-like virus which causes pseudorabies, parvovirus. encephalomyocarditus virus, a virus which causes swine vesicular disease. porcine poliovirus (techen), a virus which causes hemmaglntin~ting encephalomyocarditus, cytomegalovirus, suipoxvirus, swine influenza 25 type A, adenovirus, tr~n.~mi~ible gastroenteritus virus a virus which causes bovine viral diarrhea, parainfluenza virus 3, and vesicular stomatitis virus.
In one embodiment, the pigs from which pancreatic cells are isolated are essentially free from the following org~nism~: Toxoplasma? eperythrozoon, brucella, listeria, mycobacterium TB, leptospirillum, haemophilus suis. M. Hyopneumonia, a virus which 30 causes porcine respiratory reproductive syndrome~ a virus which causes rabies. a virus which causes pseudorabies, parvovirus, encephalomyocarditus virus, a virus which causes swine vesicular (li~ç:~e, porcine polio virus (techen), a virus which causes hemaggl-ltin~ting encephalomyocarditus, suipoxvirus, swine influenza type A, adenovirus, tr~n~mi~sible gastroenteritis virus, a virus which causes bovine viral diarrhea, and vesicular stomatitis 35 virus. The phrase "c~cnti~lly free from org~ni~m~ or substances which are capable of transmitting infection or disease to a xenogeneic recipient" (also referred to herein as "essentially pathogen-free") when referring to a swine fr~om which cells are isolated means that swine does not contain org~ni.~m~ or substances in an amount which transmits infection or ~isease to a xenogeneic recipient, e.g. a human. Example III provides representative. but WO 96/1279.1 PCT/US95/12877 not limiting examples of methods for selecting swine which are essentially free from various pathogens. The pancreatic cells of the invention can be isolated from embryonic or post-natal swine which are determined to be essentially free of such org~nisms. These swine are m~int~ined under suitable conditions until used as a source of pancreatic cells.Preferred gestational ages of the swine from which these cells are obtained are described in detail herein. Porcine pancreatic cells obtained from essentially pathogen-free swine can additionally be modified to reduce the immunogenicity of the c-ells following ~lministration to a xenogeneic subject as described herein.

=~ II. ~ETHODS OF THEINVENTION

A. Methods of Isolating Porcine Pancreatic Cells from l~;mbryonic Swine Other aspects of the invention include methods of isolating porcine pancreatic cells suitable for ~lministration to a xenogeneic subject. These methods typically include isolating 15 porcine pancreatic cells from a swine, e.g., an embryonic swine between about day thirty-one (31 ) and day thirty-five (35) of gestation, and optionally contacting the porcine pancreatic cells with at least one embryonic proliferating agent which promotes or induces cell proliferation in vitro or in vivo prior to introduction of the cells into a subject. Porcine pancreatic cells isolated according to the methods of the invention can be further modified as 20 described herein for introduction into a xenogeneic subject.
Methods of isolating pancreatic cells from primordial gut tissue are L~nown in the art.
For example~ solid pancreatic tissue samples can be dissected from surrounding gut tissue, e.g., by dissecting the tissue under a dissecting microscope. The cells in the pancreatic tissue sample can then dissociated by mechanical means, e.g., chopping and/or successive pipette ~S trituration. or by chemical means, e.g., by use of enzymes, such as trypsin or collagenase.
The swine which are employed in the method of the invention as a source of pancreatic cells include embryonic swine (swine fetuses), postnatal swine, pathogen-free embryonic swine.
and pathogen-free postnatal swine. If an embryonic pathogen-free swine is to be used as a source of pancreatic cells, semen from a boar which has ~een tested to be essenti~lly free of 30 disease- transmitting org~ni.sms is employed to artificially inseminate a female swine which is essentially free from such organisms. At about thirty-one (31) to about thirty-five (35) days of gestation~ a hysterectomv is performed under apl)Lo~l;ate conditions of sterility and the fetuses are thereafter removed in their individual amniotic sacs. Appropriate pancreatic cells or tissue are thereafter recovered. as described, for example. in Example I herein. under 35 appropriate conditions of sterility.
The methods of isolating porcine pancreatic cells suitable for atlministration to a xenogeneic subject can. optionally, further include one or more of the following steps:
lmini.stering the porcine pancreatic cells to a xenogeneic subject prior to formation of insulin-secreting islet-like aggreates or clusters in culture: ~lministering the porcine WO 96/1279~ PCT/US95/12877 pancreatic cells to a xenogeneic subject after the cells form insulin-secreting islet-like aggregates or clusters in culture; and ~lmini.~tering the porcine pancreatic eells to a xenogeneic subject as non-insulin-secreting cells which are eapable of dirrere~ tin~ in vivo to form insulin-secreting eells. The in vitro and in vivo eharacteristics of the porcine pancreatic cells of the invention are described in further detail herein.

B. Methods of Promoting or Inducing Proliferation of Porcine Pancreatic Cells Further aspects of the invention include methods of promoting or inducing proliferation of embryonic porcine pancreatic cells. These methods include eontaeting embryonic porcine pancreatic cells, in vitro or in vivo, with at least one embryonic proliferating agent, which promote(s) or induce(s) proliferation of the cells. The phrase "embryonic proliferating agent" is inten(led to include agents which promote or enhance the proliferation of embryonic poreine panereatie eells. Thus, embryonic proliferating agents which promote or induee proliferation of the poreine panereatie eells include substanees which increase the nurnber of times that an embryonie poreine panereatie eell multiplies or reproduees to form two eells (e.g.~ doubling time) in a given period of time. Speeific examples of growth faetors inelude PDGF and EGF and their equivalents. When the poreine panereatie eells of the invention are eontacted with both PDGF and EGF, their doubling time can be decreased by about 30-50% (e.g, 80 hours without growth factors vs. 58 hours with growth factors). It should be understood that the phrase "embryonic proliferating agent" also ineludes growth faetors for whieh embryonic poreine panereatie eells of a eertain gestational age (e.g.~ between about 31 to about 35 days) express a reeeptor. The methods of the invention allow the effieient produetion of large numbers of poreine panereatie eells for introduetion into xenogeneie subjeels. This is an important as about ten to fifty to about one hundred fifty million porcine panereatie eells are required to treat one human having a disease eharaeterized by insuffieient insulin aetivity. Aeeording to the methods of this invention, one fetal pig yields about one and a half million panereatie eells. Thus, about ten doublings (about 24 days) of these fetal panereatie cells results in a number of cells suffieient for introduction into a human subject. If several fetal pigs, e.g., a litter of fetal pigs? are used as donors of pancreatic eells, suffieient numbers of eells ean be generated for introduetion into a human reeipient in a matter of days.

C. Methods of Treating Diseases Characteri-ed by Insufficient Insulin Activit~,~ Using Porcine Pancreatic Cells Still further aspeets of the invention inelude methods of treating diseases charaeterized by insuffieient insulin aetivity in a subjeet, partieularly a human subject. These methods inelude ~(1mini~tering to a xenogeneic subject. an isolated population of non-insulin-secreting poreine panereatie cells having the ability to differentiate to form insulin-secreting cells a~er ~mini~tration to the subject. Such populations of cells are described in detail WO 96/1279~ PCT/US95112877 herein. The terms "introduction", "~lmini~tration", and "transplantation" are used interchangeably herein and refer to delivery of cells to a xenogeneic subject by a method or route which delivers the cells to a desired location. The term "treating" as used herein includes reducing or alleviating at least one adverse effect or symptom, e.g, absolute or relative insulin deficiency, fasting hyperglycemia, glycosuria, development of atherosclerosis, microangiopathy, nephropathy, and neuropathy, of diseases characterized by insufficient insulin activity. As used herein, the language "diseases characterized by insufficient insulin activity" include tli.~e~es in which there is an abnormal utilization of glucose due to abnormal insulin function. Abnormal insulin function includes anyabnormality or impairment in insulin production, e.g., expression and/or transport through cellular organelles, such as insulin deficiency resulting from, for example, loss of ~ cells as in IDDM (Type I diabetes), secretion, such as impairment of insulin secretory responses as in NIDDM (Type II diabetes), form of the insulin molecule itself, e.g., primary, secondary or tertiary structure, effects of insulin on target cells. e.g. insulin-resistance in bodily tissues, e.g., peripheral tissues, and responses of target cells to insulin. ~ee Braunwald~ E. et al. eds.
Harrison's Principles of Internal Medicine, Eleventh Edition (McGraw-Hill Book Company~
New York, 1987) pp. 1778-1797; Robbins, S.L. et al. Pathologic Basis of Disease, 3rd Edition (W.B. Saunders Company, Philadelphia, 1984) p. 972 for further descriptions of abnormal insulin activity in IDDM and NIDDM and other forms of diabetes.
The porcine pancreatic cells are ~flmini~tered to the subject by any appropriate route which results in delivery of the cells to a desired location in the subject where the cells can proliferate and secrete a pancreatic hormone, e.g., insulin, or enzyme. Preferred locations for pancreatic cell ~lmini~tration include those which rapidly vascularize. Common methods of iq~lmini~tering pancreatic cells to subjects, particularly human subjects~ include implantation of cells in a pouch of omentum (Yoneda, K. et al. (1989) Diabetes 38 (Suppl. 1):213-216)~
intraperitoneal injection of the cells, (Wahoff, D.C. et al. (1994) Transplant. Proc. 26:804), implantation of the cells under the kidney capsule of the subject (See, e.g, Liu~ X. et al.
(1991) Diabeles 40:858-866; Korsgren, O. et al. (1988) Transpiantation 45(3):509-514;
Simeonovic~ D.J. et al. (1982) Aust. J. Exp. Biol. Med. Sci. 60:383), and intravenous injection of the cells into. for example. the portal vein (Braesch~ M.K. et al. (1992) Transplant. Proc.
24(2):679-680: Groth, C.G. et al. (1992) Transplant. Proc. 24(3):972-973). To facilitate transplantation of the pancreatic cells under the kidney capsule~ the cells can be embedded in a plasma clot prepared from, e.g., plasma from the recipient subject (Simeonovic~ D.J. et al.
(1982) Aust. J. Exp. Biol. Med. Sci. 60:383) or a collagen matrix. Cells can be ~tlmini.ctered in a ph~rm~ceutically acceptable carrier or diluent as described herein.
This invention further pertains to methods of treating diseases characterized byinsufficient insulin activity in a subject, particularly a human subject~ in which an isolated population of porcine pancreatic cells obtained from an embryonic pig between about day 31 and day 35 of ~estation, which form insulin-secreting islet-like aggregates or clusters in WO96/1279~ PCT~S95/12877 culture, and which secrete insulin after ~imini.stration to the subject is ~imini.stered to the subject. As described herein, pancreatic cells obtained from embryonic pigs between about day 31 and 35 can be cultured as a monolayer of adherent non-insulin secreting cells. When these cells are allowed to reach confluence, they form islet-like aggregates or clusters and begin to secrete pancreatic horrnones, such as insulin, glucagon, and somatostatin, and enzymes. At this point, such aggregates can be isolated, pooled, and a-lmini.stered to a recipient subject wherein they secrete insulin. About 100,000 to 500,000 aggregates, each of which contains about 300 to 500 cells. can be used to treat one human. This number of cells can be generated from one pig fetus after about ten doublings (about 24 days) or from a litter (6-10) of fetal pigs after only a few days of doubling (about 2-10 days). Additional porcine pancreatic cells and isolated populations of such cells which can be ~tlministered to a xenogeneic subject according to this method include embryonic porcine pancreatic cells, embryonic porcine pancreatic cells obtained from an essentially pathogen-free pig, modified porcine pancreatic cells, modified porcine pancreatic cells obtained from an essçnti~lly pathogen-free pig, modified embryonic porcine pancreatic cells, and modified embryonic porcine pancreatic cells obtained fiom an essen[ially pathogen-free pig. These and other porcine pancreatic cells are described in detail herein.
The porcine pancreatic cells of the invention can be ~lmini.stered to a xenogeneic subject having a disease characterized by insufficient insulin activity in combination with 20 7~1~1mini stration of an agent which inhibits T cell activity in the subject. As used herein, an agent which inhibits T cell activity is defined as an agent which results in removal (e.g., sequestration) or destruction of T cells within a subject or inhibits T cell functions within the subject (i.e., T cells may still be present in the subject but are in a non-functional state, such that they are unable to proliferate or elicit or perform effector functions, e.g. cytokine 25 production, cytotoxicity etc.). The term "T cell" encompasses mature peripheral blood T
Iymphocytes. The agent which inhibits T cell activity may also inhibit the activity or maturation of imm~tllre T cells (e.g., thymocytes).
A preferred agent for use in inhibiting T cell activitv in a recipient subject is an immunosuppressive drug. The term "immunosuppressive drug or agent" is intended to 30 include pharmaceutical agents which inhibit or interfere with normal immune function. A
preferred immunsuppressive drug is cyclosporin A. Other immunosuppressive drugs which can be used include FK506, and RS-61443. In one embodiment, the immunosuppressive drug is ~imini.stered in conjunction with at leasl one other therapeutic agent. Additional therapeutic agents which can be ~tlministered include steroids (e.g., glucocorticoids such as 35 prednisone, methyl prednisolone and dexamethasone) and chemotherapeutic agents (e.g., azathioprine and cyclosphosphamide). In another embodiment, an immunosuppressive drug is ~iministered in conjunction with both a steroid and a chemotherapeutic agent. Suitable immunosuppressive drugs are commercially available (e.g.. cvclosporin A is available from Sandoz, Corp.. E~ast Hanover~ NJ).

WO 96/1279~ PCT/I~S95/12877 An immlm~u~ es~ive drug is ~(1mini~tered in a formulation which is compatible with the route of ~lmini~tration. Suitable routes of ~lmini.stration include intravenous injection (either as a single infusion, multiple infusions or as an intravenous drip over time), intraperitoneal injection, intramuscular injection and oral ~lmini~tration. For intravenous 5 injection, the drug can be dissolved in a physiologically acceptable carrier or diluent (e.g., a buffered saline solution) which is sterile and allows for syringability. Dispersions of drugs can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Convenient routes of ~mini~tration and carriers for immunsu~les~ive drugs are known in the art. For example, cyclosporin A can be ~lmini~tered intravenously in a saline solution. or 10 orally~ lap~ oneally or intramuscularly in olive oil or other suitable carrier or diluent.
An immunosuppressive drug is a(lmini~tered to a recipient subject at a dosage sufficient to achieve the desired therapeutic effect ~e.g., inhibition of rejection of transplanted cells). Dosage ranges for immunosuppressive drugs, and other agents which can beco~-lmini~tered therewith (e.g.. sleroids and chemotherapeutic a~ents). are known in the art (See e.~;., Freed et al. New Engl. J. Med. (1992) 327:1549: Spencer et al. (1992) Ne~ Engl. J.
Mecl. 327:1541; Widneretal. (1992)NewEngl. J. Med. 327:1556; Lindvall etal. (1992)Ann.
Neurol. 31:155; and Lindvall et al. (1992) Arch. Neurol. 46:615). A preferred dosage range for immunosl.l,ples~ive drugs, suitable for treatment of humans, is about 1-30 mg/kg of body weight per day. A preferred dosage range for cycLosporin A is about l-10 mg/kg of body weight per day, more preferably about 1-5 mg/kg of body weight per day. Dosages can be adjusted to m~int~in an optimal level of the immuno~u~l,les~ e drug in the serum of the recipient subject. For example. dosages can be adjusted to m~int~in a preferred serum level for cyclosporin A in a human subject of about 100-200 ng/ml. Il is to be noted that dosage values may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted over time to provide the optimum therapeutic response according to the individual need and the professional judgment of the person ~lmini~tering or supervising the :~rlmini~tration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
In one embodiment of the invention. an immun~.lpples~ive drug is ~-lmini~tered to a subject transiently for a sufficient time to induce tolerance to the transplanted cells in the subject. Transient ~lmini~tration of an immunosuppressive drug has been found to induce long-term graft-specific tolerance in a graft recipient (See Brunson et al. (1991) Transplantation 52:545; Hutchinson et al. (1981) Transplantation 32: 10; Green et al. (1979) Lancet 2: 123; Hall et al. (1985) J. Exp. Med. 162: 1683). Administration of the drug to the subject can begin prior to Iransplantation of the cells into the subject. For example. initiation of drug ~lmini~tration can be a few days (e.g., one to three days) before transplantation.
Alternatively drug ~tlmini~tration can begin the day of transplantation or a few days ~generally not more than three days) after transplantation. Adminislration of the drug is CA 02203032 lgg7-o4-l7 continllecl for sufficient time to induce donor cell-specific tolerance in the recipient such that donor cells will continue to be accepted by the recipient when drug ~lministration ceases.
For example~ the drug can be ~lmini~tered for as short as three days or as long as three months following transplantation. Typically, the drug is ~flmini~tered for at least one week 5 but not more than one month following transplantation. Induction of tolerance to the transplanted cells in a subject is indicated by the continued acceptance of the transplanted cells after ~lmini~tration of the immunosuppressive drug has ceased. Acceptance of transplanted tissue can be deterrnined morphologically (e.g., with skin grafts by e~minin~;
the transplanted tissue or by biopsy) or by ~ses~ment of the functional activity of the graft.
Another type of agent which can be used to inhibit T cell activity in a subject is an antibody, or fragment or derivative thereof, which depletes or sequesters T cells in a recipient. Antibodies which are capable of depleting or sequestering T cells in vivo when ~tlmini~tered to a subject are known in the art. Typically, these antibodies bind to an antigen on the surface of a T cell. Polyclonal antisera can be used, for example anti-lymphocyte serum. Alternatively, one or more monoclonal antibodies can be used. Preferred T cell-depleting antibodies include monoclonal antibodies which bind to CD2, CD3, CD4 or CD8 on the surface of T cells. Antibodies which bind to these antigens are known in the art and are commercially available (e.g., from American Type Culture Collection). A preferred monoclonal antibody for binding to CD3 on human T cells is OKT3 (ATCC CRL 8001).The binding of an antibody to surface antigens on a T cell can facilitate sequestration of T
cells in a subject andior destruction of T cells in a subject by endogenous mech~ni~m~
Alternatively. a T cell-depleting antibody which binds tO an antigen on a T cell surface can be conjugated to a toxin (e.g.~ ricin) or other cytotoxic molecule (e.g., a radioactive isotope) to facilitate destruction of T cells upon binding of the antibody to the T cells. See U.S. Patent Application Serial No.: 08/220,724, filed March 31, 1994, for further details concerning the generation of antibodies which can be used in the present invention.
Another type of antibody which can be used to inhibit T cell activity in a recipient subject is an antibody which inhibits T cell proliferation. For example~ an antibody directed against a T cell growth factor~ such as IL-2, or a T cell growth factor receptor, such as the IL-2 receptor. can inhibit proliferation of T cells (See e.g.~ DeSilva, D.R. et al. (1991) J.
Immunol. 147:3261-3267). Accordingly, an IL-2 or an IL-2 receptor antibody can be lmini~tered to a recipient to inhibi~ rejection of a transplanted cell (see e.g. Wood et al.
(1992) Neuroscience 49:410). Additionally, both an IL-2 and an IL-2 receptor antibody can be co~lmini~tered to inhibit T cell activity or can be ~tlmini.~tered with another antibody (e.g., which binds to a surface antigen on T cells).
An antibody which depLetes, sequesters or inhibits T cells within a recipient can be ~lmini.~tered at a dose and for an a~,op,iate time to inhibit rejection of cells upon transplantation. Antibodies are preferably ~-lmini~tered intravenously in a pharmaceutically acceptable carrier or diluent (e.g.~ a sterile saline solution!. Antibody ~mini.stration can WO 9611279~ PCT/I~S95112877 begin prior to transplantation (e.g., one to five days prior to transplantation) and can continue on a daily basis after transplantation to achieve the desired effect (e.g., up to fourteen days after Iransplantation). A preferred dosage range for ~tiministration of an antibody to a human subject is about 0.1-0.3 mg/kg of body weight per day. Alternatively, a single high dose of S antibody (e.g., a bolus at a dosage of about 10 mg/kg of body weight) can be ~tlmini~tered to a human subject on the day of introduction of the pancreatic cells into the subject. The effectiveness of antibody treatment in depleting T cells from the peripheral blood can be determined by comparing T cell counts in blood samples taken from the subject before and after antibody treatment. Dosage regimes can be adjusted over time to provide the optimum therapeutic response according to the individual need and the professional judgment of the person ~rlmini~tering or supervising the ~qtlmini~tration of the compositions. Dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
To assess their therapeutic potential. the porcine pancreatic cells of the învention can be introduced into exisling animal models for diabetes. These models mclude. for example.
mice in which diabetes is induced by, for example~ intravenous injection of alloxan (Korsgren, O. et al. (1993) Szlrge)y 113:205-214) or ~lmini~tration of streptozocin (Liu. X et al. (1991) Diabetes 858-866). Other animal models of diabetes~include db/db and ob/ob mouse lines (Pittman et al. (1994) Transplant. Proc. 26: 1135-1137). Therapeutic efficacy in these models of diabetes can be predictive of therapeutic efficacy in humans. Groth, C.G. et al. (1992) Transplant. Proc. 24(3):972-973. The therapeutic efficacy of the ~t1mini.~tered porcine pancreatic cells is typically determined by, for example, measurement of blood glucose concentrations using. for example. an intravenous glucose tolerance test, before and after ~(lmini~tration of the porcine pancreatic cells. Norm~ tion of hyperglycemia demonstrates that the ~rlmini~tered porcine pancreatic cells can be used to treat diseases characterized by insufficient insulin secretion. Other methods of determining the therapeutic potential are histological ea~min~lion of the pancreatic cell graft (via a biopsy), e.g., by staining for insulin, and measurement of insulin levels in blood by? for example, radioimmunoassay .
This invention is further illustrated by the following examples which in no way should be construed as being further limiting. The contents of all cited references (including literature references, issued patents, published patent applications. and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

-WO 96/1279~1 PCT/US95/12877 _ 19 _ EXAMPLES

EXAMPLE I: DISSECTION OF PANCREATIC CELLS FROM EMB~YONIC
SWINE
s Female pigs were insemin~tecl 31-35 days prior to e-lth:~ni7~tion and removal of uterus. After uterus was surgically removed and transported to sterile laboratory facilities, fetuses were delivered into a sterile dish cont~ining calcium-magnesium free phosphate buffered saline (PBS) in a horizontal laminar flow hood. Fetuses at this stage of development 10 ranged from 25-45 mm in length (crown-to-rump length).

Fetuses were removed from the storage dish cont~ining PBS and placed in a sterile dissecting dish cont~ining PBS. The dissecting dish was then placed on the stage of a dissecting microscope. With the aid of the dissecting microscope, a longitudinal incision was made down the midline of the fetus. Next~ an incision was made at right angles to the first 15 incision just below the ribs and chest cavit~. The overlying skin was pulled back to expose the internal organs of the fetus. The liver. which is the large red organ occupying virtually the entire ventral body cavity, was located and removed. After the liver was removed the stomach and small intestine were located. The stomach was white and kidney shaped at this stage of development. Once located, the stomach-was followed to the point where it joins the 20 small intestine. At the juncture between the stomach and the small intestine, the dorsal aspect of the fetal pancreas can be identified Iying in close apposition to the stomach. The fetal pancreas was the same color as the stomach and difficult to locate. Once the pancreas had been located, it was observed that the pancreas is a small tongue-like structure that was U-shaped and surrounded the posterior half of the stomach. With the fetal pancreas located it 25 was dissected away from the stomach with a pair of forceps and micro-scissors then placed in a tube cont:~ininp; sterile PBS. The procedure was repeated for all fetuses from a single donor sow.

The PBS was removed from the tube containing dissected pancreases. The tissue was then resuspended in 1.5 ml of 0.05% Trypsin~ 0.53mM EDTA and incubated at 37C for 15 30 minutes. Tissue was dissociated by triturating with a pasteur pipette until a uniform cell suspension was formed. Trypsin was stopped by adding S ml of medium (RPMI-1640 + 10%
FCS). then the cells were collected at 1000 RPM for 5 minutes at 25C. Cells were resuspended in culture media (RPMI-1640 + 10% FCS I 5 ng/ml PDGF + 100 ng/ml EGF) and plated in sterile tissue culture dishes. Cells were then allowed to adhere and grow at 37 35 C in an incubator with 5% CO ~. Figures lA-lB depict insulin staining ofthe-cultured fetal pig pancreatic cells. Figure lA is a phase micrograph of fetal pig pancreatic cells in monolayer prior to formation of islet-like clusters after two weeks of culture. No insulin staining can be detected in these cells at this time. Figure 1 B is a phase micrograph of fetal WO 96tl279-1 PCT/US95/12877 pig pancreatic cells after three weeks of culture that were allowed to form islet-like clusters (indicated by arrows). These cells stained positive for insulin.
The growth rate behavior of the pancreatic cells includes a doubling time of 80 hours without growth factors and 58 hours with growth factors. Fourteen fetuses provided S approximately 1-2 x 106 pancreatic cells. The cells can be cultured for 30 days to yield 4-8 x 109 cells. The length of time required for islet-like aggregate or cluster formation is 4-7 days after the cells reach confluence.

EXAMPLE II: INTRODUCTION OF PORCINE PANCREATIC CELLS INTO
XENOGENEIC RECIPIENTS AND DEMONSTRATION OF
INSULIN-SECRETION IN VIVO

All transplantations were done into nude mice recipients. Mice were anaesthetized by intraperitoneal ~-lmini~tration of Avertin (250mg/kg body weight) and a flank incision was made ~o expose the kidney. A small incision was made on the kidney and a small fire polished glass rod was inserted between the kidney epithelium and the kidney tissue to create a space for cells to be transplanted. Prior to transplantation, 1 o6 proliferating cells (which did not stain for insulin) were either immobilized in a rat tail collagen matrix or in a blood clot. For immobilization in rat tail collagen, 8 parts collagen at 5.29 mg/ml + 1 part 0.1 M
NaOH + 1 part 10x Hank's balanced salt solution (HBSS) were combined and the pH was adjusted to 7.4 with 0.1 M NaOH. Collagen was kept at 4C until addition to cells. Collagen did not readily gel at 4C or when pH was below 7.4, therefore, adjusting pH and warming to room temperature were essential for formation of a collagen gel. Immobilized cells were placed with forceps through the incision made on the kidney in the space previously created.
The skin incision was then closed with a wound clip and the animal transferred to a cage for recovery. In the first experiments~ 12 ~nim~ls were transplanted: 6 ~nim~l~ received cells immobilized in collagen. At one month post-transplant~ 3 animals from each group were sacrificed and the transplanted kidneys processed for histology and immunost~ining. The rem~ining ~nim~l~ are sacrif1ced at 3 months post-transplantation. For the ~nim~l~ that were sacrificed, grafts were found in 5 or 6 ~nim~ one animal inadvertently received 1/10 the number of cells inten~lç~l and that was the animal with no graft. There was no apparent difference between grafts derived from cells immobilized in blood clots or collagen. All grafts stained positive for insulin. For example, Figure 2A is a section from transplanted kidney showing the graft stained with aldehyde-fuchsin. Insulin-con~ining cells stain darkly and can be seen scattered throughout the graft. Figure 2B shows insulin staining from the same graft which is stained with a primary Mouse Insulin Ab monoclonal ~Chemicon Intl Inc.. Temecula. CA) and a secondary goat-anti-mouse antibody conjugated with horseradish peroxidase (Vector Labs Inc.. Burlingame. CA). The insulin-cont~ining cells within the graft WO 96tl279-1 PCT/US95/12877 stain more darkly than the other cells. In both figures. the insulin-positive cells are indicated by arrows, the donor graft is marked (G), and the recipient mouse kidney is mar~ed (K).

EXAMPLE III: METHODS OF PRODUCING ESSE~TIALLY PATHOGEN-FREE SWINE FROM WHICH PANCREATIC CELLS OF THE
INVENTION CAN BE OBTAINED

A. Collecting, processing, and analyzing pigfecal samples for signs of pathogensFeces are extracted from the pig's rectum manually and placed in a sterile container.
About a 1.5 cm diameter portion of the specimen was mixed thoroughly in 10 ml of 0.85%
saline. The mixture is then strained slowly through a wire mesh strainer into a 15 ml conical centrifuge tube and centrifuged at 650 x g for 2 minutes to sediment the rem~ining fecal material. The supernatant is decanted carefully so as not to dislodge the sediment. and 10%
buffered formalin was added to the 9 ml mark, followed by thorough mixing. The mixture is allowed to stand for 5 minutes. 4 ml of ethyl acetate is added to the mixture and the mixture is capped and mixed vigorously in an inverted position for 30 seconds. The cap is then removed to allow for ventilation and then replaced. The mixture is centrifuged at 500 x g for 1 minute (four layers should result: ethyl acetate, debris plug. formalin and sediment). The debris plug is rimmed using an applicator stick. The top three layers are carefully discarded by pouring them off into a solvent container. The debris attached to the sides of the tube is removed using a cotton applicator swab. The sediment is mixed in either a drop of formalin or the small amount of formalin which remains in the tube after decanting. Two separate drops are placed on a slide to which a drop of Lugol's iodine is added. Both drops are coverslipped and carefully examined for signs of pathogens, e.g., protozoan cysts of trophozoites, helminth eggs and larvae. Protozoan cyst identification is confirmed, when required, by trichrome staining.

B. Co-cultivation assay for detecting t~e presence of humaM and animal viruse~ in pig cells Materials:
C~11 lines African green monkey kidney~ (VERO), cell line American Type Culture Collection.(ATCC CCL81), human embrvonic lung fibroblasts, (MRC-5) cell line American Type Culture Collection, (ATCC CCL 171), porcine kidney, (PK-15), cell line American Type Culture Collection, (ATCC CRL 33). porcine fetal testis, (ST). cell line American Type Culture Collection, (ATCC CRL 1746).

CA 02203032 l997-04-l7 WO 96/12791 PCT/US9~/12877 Medium~ Antibiotics. and Other Cells~ and Equipment Fetal calf serum~ DMEM, Penicillin 10,000 units/ml, Streptomycin 10 mg/ml, Gentamicin 50 mg/ml, guinea pig erythrocytes, chicken erythrocytes, porcine erythrocytes, 5 Negative Control (sterile cell culture mediurn), Positive Controls: VERO and MRC-5 Cells:
Poliovirus type 1 attenuated, (ATCC VR-1 92) and Measles virus, Edmonston strain, (ATCC
VR-24), PK-1 5 and ST Cells: Swine influenza type A, (ATCC VR-99), Porcine Parvovirus, (ATCC VR-742), and Tr~n~mi~ible gastroenteritis of swine, (ATCC VR-743). Equipment:
tissue Culture Incubator, Inverted Microscope, Biological Safety Cabinet.
These materials can be used in a co-cultivation assay (a process whereby a test article is inoculated into cell lines (VERO, MRC-5, PK1 5, and ST) capable of detecting a broad range of human, porcine and other animal viruses). Hsuing, G.D., "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals" in Diagnostic Virology, 1982 (Yale University Press, New Haven, CT, 1982).
Experimental Desi~n and Methodologv:
A total of three flasks (T25) of each cell line are inoculated with at least 1 ml of test article. Three flasks of each cell line can also be inoculated with the ~prol~liate sterile cell culture medium as a negative control. Positive control viruses are inoculated into three flasks of each cell line. After an absorption period, the inoculate is removed and all flasks incubated at 35-37C for 21 days. All flasks are observed at least three times per week for the development of cytopathic effects, (CPE). of viral ori~;in. Harvests are made from any flasks inoculated with the test article that show viral CPE.
At Day 7 an aliquot of supernatant and cells from the flasks of each test article are collected and at least 1 ml is inoculated into each of three new flasks of each cell line. These subcultures are incubated at 35-37C for at least 14 days. All flasks are observed and tested as described above.
At Day 7, the flasks from each test article are also tested for viral hemadsorption.
(HAd), using guinea pig, monkey and chicken erythrocyles at 2-8C and 35-37C at 14 days postinoculation.
At Day 21, if no CPE is noted, an aliquot of supernatant from each flask is collected.
pooled. and tested for viral hemagglutination, (HA). using guinea pig. monkey, and chicken ervthrocytes at 2-8C and 35-37C. Viral identification is based on characteristic viral cytopathic effects (CPE) and reactivity in HA testing The test samples are observed for viral cytopathic effects in the following manner:
All cultures are observed for viral CPE at least three times each week for a minimum of 2 I
days incubation. Cultures are removed from the incubator and observed using an inverted microscope using at ieast 40X m~gnification. 100X or 200X m~gnification is used a~
a~lopl;ate. If any abnormalities in the cell monoiavers. including viral CPE. are noted or WO 96/1279~ PCT/US95/12877 any test articles cause total destruction of the cell monolayer, supernatant and cells are collected from the flasks and samples are subcultured in additional flasks of the same cell line. Samples can be stored at -60 to -80C until subcultured. After 7 and 14 days incubation, two blind passages are made of each test article by collecting supern~t~nt and 5 cells from all flasks inoculated with each sample. Samples can be stored at -60 to -80C
until subcultured.
Hemadsorbing viruses are detected by the following procedure: after 21 days of incubation, a hemadsorption test is performed on the cells to detect the presence of hemadsorbing viruses. The cells are washed 1-2 times with approximately 5 mls of PBS.
10 One to two mls of the ap~lol,liate erythrocyte suspension (either guinea pig, porcine, or chicken erythrocytes), prepared as described below, is then added to each Ilask. The flasks are then incubated at 2-8C for 15-20 minutes, after which time the unabsorbed erythrocytes are removed by ~hzlking the flasks. The erythrocytes are observed by placing the flasks on the lowered stage of a lab microscope and viewing them under low power m~nification. A
15 negative result is indicated by a iack of erythrocytes adhering to the cell monolayer. A
positive result is indicated by the adsorption of the erythrocytes to the cell monolayer.
Hemagglutination testing, described in detail below, is also performed after 21 days of incubation of the subcultures. Viral isolates are identified based on the cell line where growth was noted, the characteristics of the viral CPE, the hemadsorption reaction, and20 hemagglutination reactions, as applo~l;ate. The test article is considered negative for the presence of a viral agent, if any of the cell lines used in the study demonstrate viral, CPE, HA~ or HAd in a valid assay.

C. Procedure for preparing and maintaining cell lines used to detect viruses in pig cell.s Materials:
Fetal calf serum (FCS). DMEM, Penicillin 10.000 unit/ml, Streptomycin 10 mgiml, Gentamicin 50 mg/ml, T25 tissue culture flasks, tissue culture incubator (5% CO2, 37C) 30 Procedure:
Aseptic techniques are followed when performing inoculations and transfers. All inoculations and transfers are performed in a biological safety cabinet. Media is prepared by adding 10% FCS for initial seeding, 5% FCS for maintenance of cultures. as well as 5.0 ml of penicillin/streptomycin and 0.5 ml of gentamicin per 50V ml media. Sufficient media is 35 added to cover the bottom of a T25 tissue culture flask. The flask is seeded with the desired cell line and incubated at 37C. 5% C2 until cells are 80 to 100% confluent. The flasks are then inoculated with virus (QCP25).

WO 96/1279~ PCT/US95/12877 D. Preparation of erythrocyte (rbc) suspensions used in hemadsorption (HAd) and hemagglutination (HA) virus detection testing Materials:
Phosphate buffered saline, (PBS), pH 7.2, guinea pig erythrocytes stock solution, porcine erythrocytes stock solution, chicken erythrocytes stock solution, sterile, disposable centrifuge tubes, 15 or 50 ml Laboratory centrifugé

Procedure:=
An app,o~l;ate amount of erythrocytes (rbc) is obtained from stock solution. Theerythrocytes are washed 3 times with PBS by centrifugation at approximately 1000 x g for 10 minutes. A 10% suspension is prepared by adding 9 parts of PBS to each one part of packed erythrocytes. The 10% rcb suspensions are stored at 2-8C for no more than one week. 0.5%
ecb suspensions are prepared by adding 19 parts of PBS to each one part of 10% rbc suspension. Fresh 0 5% rbc suspensions are prepared prior to each day's testing.
Hemagglutination (HA) test A hemagglutination test is a test that detects viruses with the property to agglutinate erythrocytes, such as swine influenza type A, parainfluenza~ and encephalomyocarditus viruses, in the test articie. Hsuing~ G.D. (1982) Diagnostic Virology (Yale University Press, New Haven, CT);. Stites~ Daniel P and Terr, Abba I, (1991)~ Basic and Clinical Immunology (Appleton & Lange, East Norwalk, CT).

Materials:
Supernatants from flasks of the VERO cell line, MRC-5 inoculated with the test article~ flasks of positive and ne~ative controls, phosphate buffered saline (PBS), pH 7.2, guinea pig erythrocytes (GPRBC), 0.5% suspension in PBS. chicken erythrocytes (CRBC), 0.5% suspension in PBS, porcine erythrocytes (MRBC)~ 0.5% suspension in PBS
Procedure:
All sample collection and testing is performed in an approved biological safety cabinet. 0.5% suspensions of each type of erythrocytes are prepared as described above. The HA test on all cell lines inoculated with samples of the test articles at least 14 days post-inoculation. Positive and negative control cultures are included for each sample and monolayers are examined to ensure that they are intact prior to collecting samples.
At least 1 ml of culture fluid from each flask inoculated with the test article is collected and pooled. 1 ml samples from the negative and positive control cultures are also collected and pooled. A set of tubes is labeled with the sample number and Iype of erythrocyte (distinguish positive and negative suspension) to be added. Racks may be labeled to differentiate the type of erythrocyte. 0.1 ml of sarnple is added to each tube. 0.1 ml of the ~lo~l;ate erythrocyte suspension is added to each tube. Each tube is covered with parafilm and mixed thoroughly. One set of tubes is incubated at 2-8C until tight buttons form in the negative control in about 30-60 minutes. Another set of tubes is incubated at 35-37C until tight buttons forrn in the negative control in about 30-60 minutes.
Formation of a tight button of erythrocytes indicates a negative result. A coating of the bottom of the tube with the erythrocytes indicates a positive result.

0 E. Methods usedforfluorescent antibodystain of cell suspensions obtainedJ~om flasks used in detection of viruses in porcine cells using cell culture techniques (as described in Sections B and C) Materials:
Pseudorabies, parvovirus, enterovirus. adenovirus~ tr~n~mi~ible Gastroenteritis Virus.
bovine viral diarrhea~ encephalomyocarditus virus, parainfluenza, vesicular stomatitis virus.
microscope slides, PBS, incubator with humidifying chamber at 36C~ Evan's blue coutner stain, DI Water~ fluorescent microscope, trypsin~ serum cont~ining media~ acetone, T25 Flask.
Procedure:
Cells (described in Sections B and C) are trypsinized to detach them from the T25 flask and sufficient media is added to neutralize trypsin activity. A drop of cell suspension is placed on each microscope slide and allowed to air dry. A slide for each fluorescent antibody is prepared. Cells are fixed by immersion in acetone for five minutes. Each fluorescent antibody solution is placed on each slide to cover cells and the slides are incubated in humidifying chamber in incubator at 36C for 30 minutes. The slides are then washed in PBS for five minutes. The wash is repeated in fresh PBS for five minutes followed by a rinse with DI water.
The cells are counterstained by placing Evan's blue solution on each slide to cover cells for five minutes at room temperature. The slides are then washed in PBS for five minlltes. The wash is repeated in fresh PBS for five minutes followed by a rinse with DI
water. The slides are then allowed to air dry. Each slide is inspected under a fluorescent microscope. Any fluorescent inciusion bodies characteristic of infection are considered a positive result for the presence of virus.
-F. Procedures for Defining Bacteremic Pigs Materials:
Anaerobic BMB agar (5% sheep blood, vitamin K and hemin [BMB/blood]), 5 chocolate Agar with Iso Vitalex~ Sabaroud dextrose agar/Emmons, 70% isopropyl alcohol swabs, betadine solution, 5% CO2 incubator at 35-37C~ anaerobic blood agar plate, gram stain reagents (Columbia Broth Media), aerobic blood culture media (anaerobic brain heart infusion with vitarnin K& hemin), septicheck media system, vitek bacterial identification system~ laminar flow hood, microscope, and bacteroids and Bacillus stocks : -~
Procedure:
Under a larninar flow hood, disinfect the tops of bottles for aerobic and anaerobic blood cultures of blood obtained from pig with 70% isopropyl alcohoL then with betadine The rubber stopper and cap from the aerobic blood culture bottle are removed and a renal 15 septicheck media system is altached to the bottie. The bottles are incubated in 5% CO, for 21 days at 35-37C, and observed daily for any signs of bacterial growth (i.e. gas bubbles~
turbidity, discoloration or discrete clumps). Negative controls consisting of 5cc of sterile saline in each bottle and positive controls consisting of Bacillus subtilis in the aerobic bottle and Bacteriodes Vulgaris in the anaerobic bottle are used. If signs of bacterial growth are 20 observed~ a Gram stain is prepared and viewed microscopically at I OOx oil immersion for the presence of any bacteria or fungi. The positive bottles are then subcultured onto both chocolate agar plates with Iso Vitlex and onto BMB plates. The chocolate plate is incubated at 35-37C in 5% C02 for 24 hours and the BMB anaerobically at 35-37C for 48 hours. Any yeast or fungi that is in evidence at gram stain is subcultured onto a Sabaroud 25 dextrose/Emmons plate. The Vitek automated system is used to identify bacteria and yeast.
Fungi are identified via their macroscopic and microscopic characteristic. If no signs of growth are observed at the end of 21 days~ gram stain is prepared and observed microscopically for the presence of bacteria and fungi.
Absence of growth in the negative control bottles and presence of growth in the 30 positive control bottles indicates a valid test. The absence of any signs of growth in both the aerobic and anaerobic blood culture bottles~ as well as no org~ni~m~ seen on gram stain indicates a negative blood culture. The presence and identification of microorganism(s) in either the aerobic or anaerobic blood culture bottle indicates of a positive blood culture: this typicall is due to a bacteremic state.
Equivalents Those skilled in the art will recognize. or be able to ascertain using no more than routine experimentation. manv equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (64)

1. An isolated non-insulin-secreting porcine pancreatic cell having the ability to differentiate into an insulin-secreting cell upon introduction into a xenogeneic subject.

2. The isolated non-insulin-secreting porcine pancreatic cell of claim 1, further having the ability to produce glucagon prior to introduction into the xenogeneic subject.

3. The isolated non-insulin-secreting porcine pancreatic cell of claim 1, further having the ability to produce somatostatin prior to introduction into the xenogeneic subject.

4. The isolated non-insulin-secreting porcine pancreatic cell of claim 1, which is obtained from an embryonic pig.

5. The isolated non-insulin-secreting porcine pancreatic cell of claim 4, which is obtained from an embryonic pig between about day 31 and about day 35 of gestation.

6. An isolated population of non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject.

7. The isolated population of non-insulin-secreting porcine pancreatic cells of claim 6, which are obtained from an embryonic pig.

8. The isolated population of non-insulin-secreting porcine pancreatic cells of claim 7, which are obtained from an embryonic pig between about day 31 and about day 35 of gestation.

9. A cell culture comprising a population of non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject and a medium suitable to support the growth of the cells.

10. The cell culture of claim 9, wherein the cells are obtained from an embryonic pig between about day 31 and about day 35 of gestation.

11. A porcine pancreatic cell, which, in unmodified form, has at least one antigen on the cell surface which is capable of stimulating an immune response against the cell in a xenogeneic subject, wherein the antigen on the cell surface is altered to inhibit rejection of the cell when introduced into the xenogeneic subject.

12. The porcine pancreatic cell of claim 11, wherein the antigen on the cell surface which is altered is an MHC class I antigen.

13. The porcine pancreatic cell of claim 12, which is contacted prior to introduction into a xenogeneic subject with at least one MHC class I antibody, or fragment or derivative thereof, which binds to the MHC class I antigen on the cell surface but does not activate complement or induce lysis of the cell.

14. The porcine pancreatic cell of claim 13, wherein the MHC class I antibody isan anti-MHC class I F(ab')2 fragment.

15. The porcine pancreatic cell of claim 14, wherein the MHC class I F(ab')2 fragment is a F(ab')2 fragment of a monoclonal antibody PT85.

16. The porcine pancreatic cell of claim 11, which is obtained from an embryonicpig.

17. The porcine pancreatic cell of claim 16, which is obtained from an embryonicpig between about day 31 and about day 35 of gestation.

18. An isolated population of porcine pancreatic cells which, in unmodified form, have at least one antigen on the surface of the cells which is capable of stimulating an immune response against the cells in a xenogeneic subject wherein the antigen on the surface of the cells is altered to inhibit rejection of the cells when introduced into the xenogeneic subject.

19. The isolated population of porcine pancreatic cells of claim 18, wherein thecells are in the form of islet-like aggregates.

20. The isolated population of porcine pancreatic cells of claim 19, which are obtained from an embryonic pig.

21. The isolated population of porcine pancreatic cells of claim 20, which are obtained from an embryonic pig between about day 31 and about day 35 of gestation.

22. An isolated pancreatic cell obtained from a pig which is essentially free from organisms or substances which are capable of transmitting infection or disease to a xenogeneic recipient of the cells.

23. The porcine pancreatic cell of claim 22, which is isolated from a pig which is essentially free from at least one organism selected from the group consisting of parasites, bacteria, mycoplasma, and viruses.

24. The porcine pancreatic cell of claim 23, which is obtained from an embryonicpig.

25. The porcine pancreatic cell of claim 24, which is obtained from an embryonicpig between about day 31 and about day 35 of gestation.

26. An isolated population of pancreatic cells obtained from a pig which is essentially free from organisms or substances which are capable of transmitting infection or disease to a xenogeneic recipient of the cells.

27. The isolated population of pancreatic cells of claim 26, wherein the cells are in the form of islet-like aggregates.

28. A method of promoting proliferation of embryonic porcine pancreatic cells, comprising contacting the cells with an embryonic proliferating agent which promotes proliferation of the cells.

29. The method of claim 28, wherein embryonic porcine pancreatic cells are in the form of islet-like aggregates.

30. The method of claim 28, wherein the cells are embryonic porcine pancreatic cells between about day 31 and about day 35 of gestation.

31. The method of claim 28, wherein the embryonic proliferating agent is a growth factor for which the embryonic porcine pancreatic cells express a receptor.

32. The method of claim 28, wherein the embryonic proliferating agent is platelet-derived growth factor.

33. The method of claim 28, wherein the embryonic proliferating agent is epidermal growth factor.

34. A method of isolating and promoting proliferation of porcine pancreatic cells in vitro prior to administration of the cells to a xenogeneic subject, comprising a) isolating non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject from an embryonic pig; and b) contacting the non-insulin-secreting porcine pancreatic cells with an embryonic proliferating agent which promotes proliferation of the cells.

35. The method of claim 34, wherein the non-insulin-secreting porcine pancreaticcells are isolated from an embryonic pig between about day 31 and about day 35 of gestation.

36. The method of claim 34, further comprising administering the non-insulin-secreting porcine pancreatic cells to a xenogeneic subject prior to formation of insulin-secreting islet-like aggregates in vitro.

37. The method of claim 34, further allowing the non-insulin-secreting porcine pancreatic cells to form insulin-secreting islet-like aggregates in vitro and administering the insulin-secreting islet-like aggregates to a xenogeneic subject.

38. A method of treating a disease characterized by insufficient insulin activity in a subject, comprising administering to a subject having the disease an isolated population of non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject.

39. The method of claim 38, wherein the non-insulin-secreting porcine pancreaticcells are obtained from an embryonic pig.

40. The method of claim 39, wherein the non-insulin-secreting porcine pancreaticcells are obtained from an embryonic pig between about day 31 and about day 35 of gestation.

41. The method of claim 38, wherein the subject is a human.

42. The method of claim 41, wherein the disease is Type II diabetes.

43. A method of treating a disease characterized by insufficient insulin activity in a subject, comprising administering to a subject having the disease an isolated population of porcine pancreatic cells obtained from an embryonic pig between about day 31 and day 35 of gestation, the cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject.

44. A method of treating a disease characterized by insufficient insulin activity in a subject, comprising administering to the subject an isolated population of porcine pancreatic cells, which cells, in unmodified form, have at least one antigen on the surface of the cells which is capable of stimulating an immune response against the cells in a xenogeneic subject, wherein the antigen on the surface of the cells is altered to inhibit rejection of the cells when administered to the subject.

45. The method of claim 44, wherein the porcine pancreatic cells are in the formof islet-like aggregates.

46. A method of treating a disease characterized by insufficient insulin activity in a subject, comprising administering to a subject having the disease an isolated population of porcine pancreatic cells obtained from a pig which is essentially free from organisms and substances which are capable of transmitting infection or disease to a xenogeneic recipient of the cells.

47. The method of claim 46, wherein the porcine pancreatic cells are obtained from a pig which is essentially free from at least one organism selected from the group consisting of parasites, bacteria, mycoplasma, and viruses.

48. The method of claim 46, wherein the porcine pancreatic cells are in the formof islet-like aggregates.

49. The method of claim 46, wherein the porcine pancreatic cells are obtained from an embryonic pig.

50. The method of claim 49, wherein the porcine pancreatic cells are obtained from an embryonic pig between about day 31 and about day 35 of gestation.

51. A method of treating a disease characterized by insufficient insulin activity in a subject, comprising administering to the subject an isolated population of non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject, which cells, in unmodified form, have at least one antigen on the surface of the cells which is capable of stimulating an immune response against the cells in a xenogeneic subject, wherein the antigen on the surface of the cells is altered to inhibit rejection of the cells when administered to the subject.

52. The method of claim 51, further comprising administering an immunosuppressive agent to the subject.

53. A delivery device having a needle, the device containing non-insulin-secreting porcine pancreatic cells having the ability to differentiate into insulin-secreting cells upon introduction into a xenogeneic subject.

54. The delivery device of claim 53 which is a syringe.

55. The delivery device of claim 53, wherein the non-insulin-secreting porcine pancreatic cells are obtained from an embryonic pig.

56. The delivery device of claim 55, wherein the non-insulin-secreting porcine pancreatic cells are obtained from an embryonic pig between about day 31 and about day 35 of gestation.

57. The delivery device of claim 53, wherein the cells are suspended in a solution.

58. The delivery device of claim 53, wherein the cells are embedded in a supportmatrix.

59. A delivery device having a needle, the device containing embryonic porcine pancreatic cells obtained from an embryonic pig between about day 31 and about day 35 of gestation.

60. The delivery device of claim 59, wherein the embryonic porcine pancreatic cells are in the form of islet-like aggregates.

61. A delivery device having a needle, the device containing porcine pancreatic cells which, in unmodified form, have at least one antigen on the cell surface which is capable of stimulating an immune response against the cells in a xenogeneic subject, wherein the antigen on the cell surface is altered to inhibit rejection of the cell when introduced into the xenogeneic subject.

62. The delivery device of claim 61, wherein the porcine pancreatic cells are in the form of islet-like aggregates.

63. A delivery device having a needle, the device containing porcine pancreatic cells obtained from a pig which is essentially free from organisms or substances which are capable of transmitting infection or disease to a xenogeneic recipient of the cells.

64. The delivery device of claim 63, wherein the porcine pancreatic cells are in the form of islet-like aggregates.