JP2003513624A - Medium for preparing dedifferentiated cells - Google Patents

Abstract

  (57) [Summary] The present invention relates to a medium for preparing dedifferentiated cells derived from postnatal islets of Langerhans. The medium is for generating, retaining, and growing an effective amount of a solid matrix environment for three-dimensional culture, a soluble matrix protein, and the dedifferentiated intermediate cells in a physiologically acceptable culture medium. The first and second factors are included. Such a medium may be used in an in vitro method for the growth of islet cells.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION (a) Field of the Invention The present invention relates to a medium for preparing dedifferentiated cells, and more particularly to at least biology, which may be used in in vitro methods for the proliferation of islet cells. It relates to a basal feeding medium for the development, maintenance and growth of a dedifferentiated cell population with the above possibilities.

(B) Description of the Prior Art Diabetes mellitus has been classified into type I, or insulin-dependent diabetes mellitus (IDDM) and type II, or non-insulin-dependent diabetes mellitus (NIDDM). NIDDM patients also have (a) non-obese (probably evolutionary IDDM)
), (B) obesity, and (c) adult-onset diabetes (younger patients).
Of the population with diabetes mellitus, about 20% have IDDM. Diabetes occurs either when a decrease in insulin production occurs or when a reduced sensitivity to insulin cannot be guaranteed by the increased ability of insulin secretion. In patients with IDDM, decreased insulin secretion is a major factor in pathology, while in patients with NIDDM, decreased insulin sensitivity is a major factor. The mainstay of treatment of diabetes, especially type I diabetes, has been the administration of exogenous insulin.

Principles for a More Physiological Treatment Tight glucose control appears to be the key for prevention of secondary complications of diabetes. The results of the Diabetes Complications and Controls Trial (DCCT), a randomized, multi-institutional trial of 1441 patients with insulin-dependent diabetes, showed diabetic retinopathy, nephropathy, and neuropathy. It was shown that the onset and progression could be delayed by intensive insulin treatment (The Diabetes Control).
and Complication Trial Research Gro
up, N.N. Engl. J. Med. , 1993; 29: 977-986). Tight glucose control, however, was associated with a 3-fold increase in the incidence of severe hypoglycemia, including episodes of seizures and coma. Moreover, glycosylated hemoglobin levels were reduced in the treatment group, but despite the enormous amount of effort and financial resources allocated to support patients for intensive care, only 5% <6.05%. Did not maintain average levels (The Diabetes C
on control and Complication Trial Real
ch Group, N.C. Engl. J. Med. , 1993; 29: 977-9.
86). DCCT results showed that intensive control of glucose could significantly reduce (but not completely protect) long-term microvascular complications of diabetes mellitus.

Other Treatment Options The delivery of insulin in a physiological manner is such that insulin is first Banti.
It is the ultimate goal since it was purified by ng, Best, McLeod and Collip. Even in patients undergoing tight glucose control, however, exogenous insulin achieves glucose metabolism of the endogenous insulin source in response to momentary changes in glucose concentration, thus leading to the development of microvascular complication levels over a long period of time. Could not defend against.

A major goal of diabetic research has thus been the development of new forms of treatment that endeavor to exactly reproduce normal physiological conditions. One such approach, a closed-circuit insulin pump coupled to a glucose sensor,
We have mimicked β-cell function in that insulin secretion is tightly regulated, but have not yet succeeded. Only total endocrine replacement therapy in the form of implants has proven effective in treating diabetes mellitus. Transplantation of insulin-producing tissues is a logical advance over subcutaneous insulin injection, but whether the risk of interference and the risks associated with long-term immunosuppressive treatment is lower than those of diabetic patients under conventional treatment. Still not clear at all.

Despite early evidence of the potential benefits of vascularized pancreas transplantation, complex surgical interventions remain, requiring long-term administration of systemic immune suppression with its attendant side effects And Moreover, almost 50% of successful transplant patients exhibit impaired tolerance curves (Wright FH et al., Arch. Surg., 1989:
124: 796-799; Landgraft R et al. , Diabe
tologia 1991; 34 (suppl 1): S61; Morel P.
et al. , Transplantation 1991; 51: 990-.
1000), question their protection against the long-term complications of systemic hyperglycemia.

The major complications of whole pancreas transplantation, as well as the requirement for long-term immunosuppression, limited its wide applicability and provided impetus for the development of islet transplantation. In theory, islet-only transplants allow tight glycemic control, but have some potential advantages over whole pancreas transplants. These include: (i) minimal surgical morbidity with direct injection of islets into the liver through the portal vein; (ii) simple re-implantation potential for graft failure; (iii) ) Elimination of complications associated with exocrine pancreas; (i
v) Possibly less immunogenicity of the pancreatic islets, eliminating the requirement for immunosuppression and allowing early transplantation into non-uremic diabetes; (v) to reduce their immunogenicity. Possibility of modifying islets in vitro prior to transplantation; (vi) the ability to encapsulate islets in artificial membranes to isolate them from the host immune system; and (vii) immunize as part of a biohybrid system. Xenot transplantation of isolated islets
related possibilities of using the (transplantation). Moreover, they allow banking of cryopreserved tissues of the endocrine glands prior to transplantation and allow a careful and standardized quality control program.

Problems with Islet Transplantation A sufficient number of isogenic islets transplanted at reliable transplant sites can only reverse metabolic abnormalities in short-term diabetic recipients. Hyperglycemia recurred within 3-12 months in patients who had been post-euglycemic transplanted. (Orl
off M, et al. , Transplantation 1988; 45.
: 307). The recurrence of the diabetic state over time is due to either non-regular localization of the islets, disruption of the enteroinsular axis, or transplantation of inadequate islet cell mass (Bretzel RG, et.
al. In: Bretzel RG, (ed) Diabetes mellit
us (Berlin: Springer, 1990) p. 229).

There are few studies of long-term natural growth of islet transplants that examine parameters other than graft function. Only one study was found, where special efforts were made to study the graft morphology (Alejandro R, et a.
l. , J Clin Invest 1986; 78: 1339). In that study, purified islets were transplanted into the liver of dogs through the portal vein. During postponed follow-up, delayed dysfunction of graft function occurred. Unfortunately, the grafts were only examined at the end of the study and not over the time of diminished function. Delayed graft dysfunction was also confirmed by dogs in other investigators (Warnock GL et al., Can. J. et al.
Surg. , 1988; 31: 421 and primates; Sutton R, et a.
l. , Transplant Proc. , 1987; 19: 3525). Most dysfunction is presumed to be the result of rejection without adequate immunosuppression.

Because of these dysfunctions, there is currently a great deal of enthusiasm for islet immunoisolation that can eliminate the requirement for immunosuppression. I cannot help but pay attention to the reason. Immunosuppression is detrimental to the recipient and may impair islet function and possibly cell viability (Metrakos P, et al., J.S.
urg. Res. , 1993; 54: 375). Unfortunately, microencapsulated islets injected into the peritoneal cavity of dogs fail within 6 months (
Soon-Shiong P, et al. , Transplantation
1992; 54: 769), and islets placed in the vascularized biohybrid pancreas also cease to function, but at about the first year. In each case, however, histological evaluation of the graft showed a substantial loss of islet mass in these devices (Lanza RP, et al., Diabetes 1992).
41: 1503). No reason was raised for these changes. Therefore, the retention of effective islet cell mass after transplantation remains an important problem.

In addition to this unsolved problem, the lack of source tissue for transplantation is an ongoing problem. The number of human donors is insufficient to keep up with the potential number of recipients. Furthermore, given the current state of the art of islet isolation, the number of islets that can be isolated from a single pancreas is far from that needed to effectively reverse hyperglycemia in human recipients.

In response, three technologies have been proposed and are in development. First, cold preservation of islets and banking of islets. But the techniques involved are expensive and cumbersome, and it's not easy to make them useful for a wide range of adaptations. Furthermore, islet cell mass is also reduced during the freeze-drying cycle. Thus, this is a poor long-term interpretation of the problem of inadequate islet cell mass. Second is the development of xenotransplantation of pancreatic islets. This idea has been coupled to islet encapsulation technology that produces biohybrid implants that, at least in theory, do not require immunosuppression. There are a number of problems to be addressed with this approach, especially the issue of retention of islet cell mass after transplantation remains. Third is the confidence in human fetal tissue that it should have a great ability to be grown ex vivo and then transplanted. However, in addition to the issues of limited tissue availability, immunogenicity, there are complex ethical issues surrounding their use, such as tissue source, which are not immediately understood. However,
There are almost unlimited alternatives that offer similar possibilities for cell mass growth.

A completely novel approach proposed by Rosenberg in 1995 (
Rosenberg L et al. , Cell Transplantat
Ion, 1995; 4: 371-384) was the development of techniques for controlling and modulating islet cell neogenesis and de novo islet formation both in vitro and in vivo. The above concept was that (a) the induction of islet cell differentiation was indeed controllable;
It was hypothesized that (b) implied the persistence of adult pancreatic stem cell-like cells; and (c) the signals that would drive the entire process were identified and manipulated.

In a series of in vivo studies, Rosenberg and coworkers found that these concepts are in principle valid in the in vivo setting (Rosenbe).
rg L et al. , Diabetes, 1988; 37: 334-341.
Roseenberg L et al .; , Diabetologia, 199
6; 39: 256-262), and diabetes could be completely changed.

A well-known technique for in vitro islet cell expansion from non-fetal tissue sources is Peck.
And collaborators (Corneliu JG et al., Horm. Me
tab. Res. , 1997; 29: 271-277), they describe the isolation of pluripotent stem cells from the pancreas of adult mice that can directly direct insulin-producing cells. These findings were not widely accepted. First, the results were not proven to be reproducible. Second, so-called pluripotent cells have not been well characterized with respect to phenotype. And third, the cells were not shown to be pluripotent.

More recently, two competing technologies have been engineered to use the pancreatic β-cell line (Efra
t S, Advanced Drug Delivery Reviews, 1
998; 33: 45-52), and the use of pluripotent embryonic stem cells (Shamblot).
t MJ et al. , Proc. Natl. Acad. Sci. USA, 1
998; 95: 13726-13731). The former view is fascinating but is accompanied by serious problems. Not only must the engineered cell be able to produce insulin, but it must also respond in a physiological manner to normal levels of glucose, and the glucose sensitization machinery must engineer it intracellularly. It is far from being fully understood. Many proposed cell lines are also transformed lines and thus have the potential for neoplasia. For the latter view, it is intriguing to have embryonic stem cells, as the theoretical possibility is to induce differentiation in all directions, including pancreatic β-cell differentiation. However, the signals needed to achieve this milestone remain unknown.

Insulin, a standard for the preparation of dedifferentiated intermediate product cells from postnatal Langerhans islets, their proliferation and guided induction of islet cell differentiation, which can be used for the treatment of diabetes mellitus It would often be desirable to provide a criterion for directing producer cells.

SUMMARY OF THE INVENTION One object of the invention is the standard for the preparation of dedifferentiated intermediate product cells from postnatal Langerhans islets, their proliferation and guided induction of islet cell differentiation, It would be highly desirable to provide criteria that lead to insulin-producing cells that can be used for the treatment of diabetes mellitus.

According to one aspect of the invention, there is provided an in vitro method for the proliferation of pancreatic islet cells, the steps of: a) preparing dedifferentiated cells derived from post-natal Langerhans islet cells; b) dedifferentiating as described above. And c) inducing the islet cell differentiation properties of the expanded cells of step b) into insulin-producing cells.

Preferably, step a) and step b) are achieved simultaneously using a solid phase matrix, a basal feeding medium and a suitable growth factor, thereby generating a dedifferentiated cell at least with respect to its biological capacity. , Allow retention and growth.

Such a medium for preparing dedifferentiated cells from post-natal Langerhans islets is provided in a physiologically acceptable culture medium in an effective amount of a solid phase matrix environment, matrix for three-dimensional culture. Generate protein and dedifferentiated cells,
It includes first and second factors that are maintained and propagated.

Preferably, the first factor induces an increase in intracellular cAMP and the second factor is derived from acinar cells. The acinar cells must be present in addition to the other three factors for changes to occur. The first factor is one or more cholera toxins (
CT), forskolin, high glucose concentration, promoter of cAMP, and EGF.

The culture medium is DMEM / 12 supplemented with an effective amount of fetal bovine serum, eg 10%.
May be included. Matrix proteins are one or more of laminin, collagen type I
And Matrigel®.

Preferably step c) is achieved by removing cells from the matrix and is suspended in a basal liquid medium containing soluble matrix proteins and growth factors.

Preferably, the basal liquid medium is C supplemented with at least 10% fetal bovine serum.
MRL1066, provided that the soluble matrix protein and growth factor are
It is selected from the group consisting of fibronectin, IGF-1, IGF-2, insulin, and NGF. The basal liquid medium may further comprise a glucose concentration of at least 11 mM. The basal liquid medium may further comprise inhibitors of known intracellular signaling pathways of apoptosis and / or inhibitors of p38.

According to another aspect of the invention, there is provided an in vitro method for producing cells having at least biological capacity, comprising: a) dedifferentiating from post-natal Langerhans islet cells from a patient. Prepare cells;
Thereby, when the dedifferentiated cells are introduced in situ in a patient, the cells proliferate, and by introducing the differentiation characteristics of pancreatic islet cells, they become insulin-producing cells in situ. Including.

According to another aspect of the invention, there is provided a method of in vitro expansion of stem cells, the steps of: a) preparing a dedifferentiated intermediate product cell derived from a stem cell; b) in vitro dedifferentiated intermediate product as described above. Growing the product cells; and c) inducing the in vitro stem cell differentiation characteristics of the expanded cells of step b) into stem cells.

[0028] Preferably, the stem cells are selected from the group consisting of muscle, skin, bone, cartilage, lung, liver, bone marrow and hematopoietic cells. According to another aspect of the invention, there is provided a method of treating diabetes mellitus in a patient, comprising the steps of: a) preparing dedifferentiated cells derived from postnatal Langerhans islet cells of the patient; and b) dedifferentiated cells as described above. Is introduced in situ in the patient, provided that
Proliferating said cells in situ and inducing the differentiation characteristics of pancreatic islet cells in situ to become insulin-producing cells.

According to another aspect of the invention, there is provided a method of treating diabetes mellitus in a patient, comprising the steps of: a) preparing dedifferentiated cells derived from postnatal Langerhans islet cells of the patient; and b) in vitro. And c) inducing the in vitro islet cell differentiation characteristics of the expanded cells of step b) into insulin-producing cells; and d) the cells of step c) in situ in the patient. Induce, but includes the cells producing insulin in situ.

For purposes of the present invention, the following terms are defined below. The expression "Isle of Langerhans after birth" is of any origin, for example humans,
It is intended to mean porcine and canine islet cells. DETAILED DESCRIPTION OF THE INVENTION In vivo cell transformation leading to β-cell neogenesis and de novo islet formation can be understood in the context of established concepts of evolving biology.

Transdifferentiation is the change of one differentiated phenotype to another and includes morphological and functional phenotypic markers (Okada TS., Development. Gro.
wt and Differ. 1986; 28: 213-321). An example of the best study of this process is the conversion of pigment cells in the amphibian iris to lens fibers, which progress through the pathways of cell dedifferentiation, proliferation and finally redifferentiation (
Okada TS, Cell Diff. 1983; 13: 177-183; O
kada TS, Kondoh H, Curr, Top Dev. Biol. ，
1986; 20: 1-433; Yamada T, Monogr. Dev. Bi
ol. , 1977; 13: 1-124). Direct transdifferentiation without cell division was also reported, but less common (Beresford WA, Cell Diff).
er. Dev. , 1990; 29: 81-93). It has been reported that transdifferentiation is essentially irreversible, ie transdifferentiated cells do not revert to the cell type in which they originated, which was recently reported to be untrue (Danto SI
et al. , Am. J. Respir. Cell Mol. Biol. , 1
995; 12: 497-502). Nevertheless, the proof of trans-differentiation depends on the detailed definition of the phenotype of the origin cell and on demonstrating that the new cell type is in fact inherited from the defined cell (Okada TS, Develo).
p. Growth and Differ. 1986; 28: 213-321).
.

In many instances, transdifferentiation comprises a continuous process. In the early part of the process, the intermediate cells did not appear to express the original phenotype or later differentiated cell types, and thus they were said to be dedifferentiated. The entire process involves DNA replication and cell proliferation. It is hypothesized that dedifferentiated cells are innately capable of forming either the original cell type or the novel cell type, ie pluripotency (
Itoh Y, Eguchi G, Cell Differ. , 1986; 18
173-182; Itoh Y, Eguchi G, Development. Bio
logy, 1986; 115: 353-362; Okada TS, Devel.
op. Growth and Differ, 1986; 28: 213-321.
).

The cellular phenotypic stability of an adult organism is probably related to the extracellular environment, as well as the cytoplasmic and nuclear components that interact to regulate gene expression. Cell phenotypic conversion appears to be achieved by a selective milieu of gene expression, controlling the developmental commitment of the cell's endpoint.

The pancreas consists of several types of endocrine and exocrine cells, each of which responds to various trophic effects. The potential of these cells to undergo phenotypic changes has been extensively investigated, but with implications for understanding pancreatic diseases such as cancer and diabetes mellitus. Transdifferentiation of pancreatic cells first came to the attention almost 10 years ago. Hepatocyte-like cells do not normally exist in the pancreas, and after carcinogen administration to hamsters (Rao MS
et al. , Am. J. Pathol. , 1983; 110: 89-94; S.
carpelli DG, Rao MS, Proc. Natl. Acad. Sc
i. USA 1981; 78: 2577-2581) and after feeding the rats with a copper depleted diet (Rao MS, et al., Cell Differ., 1).
986; 18: 109-117). Recently, trans-differentiation of isolated acinar cells into tube-like cells was observed by several groups (Arias).
AE, Bendayan M, Lab Invest. , 1993; 69:51.
8-530; Hall PA, Lemoine NR, J. Am. Pathol. , 1
992; 166: 97-103; Tsap MS, Duuid WP, Exp.
． Cell Res. , 1987: 168: 365-375). Given these observations, it is probably relevant that liver and pancreas primordium come from a common endothelium during embryonic development.

Although the factors controlling pancreatic growth and functional maturation of human endocrine glands during the fetal and postnatal period are poorly understood, the presence of specific factors in the pancreas has been postulated. (Picetet RL et al. In: Extracellu
lar Matrix Fluences o Gene Express
ion. Slavkin HC, Greulich RC (eds). Acad
emic Press, New York, 1975, pp. 10).

Some information is available on exocrine growth factors. Mesenchyme (MF) was extracted from a specific fraction of mid-pregnant rat or chicken embryo homogenates. MF affects cell development by interacting at the cell surface of precursor cells (Rutter WJ. The development of the ee).
ndocrine and exocrine pancreas. In: Th
e Pancreas. Fitzgerald PJ, Morson B (ed
s). Williams and Wilkins, London, 1980,
pp. 30), and thereby affects the cell types that emerge during pancreatic development (Githens S. Differentiation and dev.
element of the exocrine pancreas in
animals. In: Go VLW, et al. (Eds). The E
xocrine Pancreas: Biology, Pathobilog
y and Diseases. Raven Press, New York,
1986, pp. 21). MF consists of at least two basic components, a cardiac stabilizing component whose action can be doubled by cyclic AMP analogs, and another high molecular weight protein component (Rutter WJ, et al, In: The Panc.
reas. Fitzgerald PJ, Morson B (eds). Wil
liams and Wilkins, London, 1980, pp. 30)
. In the presence of MF, cells actively divide and often differentiate into non-endocrine cells.

Other factors are also involved in endocrine maturation. Soluble peptide growth factor (GF)
Is a group of nutrients that regulate both cell proliferation and cell differentiation. These growth factors are multifunctional and may trigger a wide range of cellular responses (Sporn & Roberts, Nature, 332: 217-1).
9, 1987). Their actions can be divided into two general categories of effects on cell proliferation, including cell growth, cell division and initiation of cell differentiation; and effects on cell function. They differ from polypeptide hormones in that they act in an autocrine and / or paracrine manner (Goustin
AS, Leof EB, et al. Cancer Res. , 46: 101
5-1029, 1986; Underwood LE, et al. , Clin
ics in Endocrinol. & Metabol. , 15: 59-7
7, 1986). The specificity of their role in individual processes, including growth, is
It needs to be elucidated.

One family of growth factors is the somatomedins. Insulin-like growth factor-I (IGF-I) is synthesized and released by β cells of fetal and neonatal rat pancreatic islets (Hill DJ, et al., Diabetes, 36: 4).
65-471, 1987; Rabinovitch A, et al. , Dia
betes, 31: 160-164, 1982; Romanus JA et.
al. , Diabetes 34: 696-792, 1985). IGF-II
Was identified in the human fetal pancreas (Bryson JM et al., J. et al.
Endocrinol. , 121: 367-373, 1989). Both of these factors enhance neonatal β-cell replication when added to the culture medium (
Hill DJ, et al. , Diabetes, 36: 465-471, 1
987; Rabinovitch A, et al. , Diabetes, 31
: 160-164, 1982). Thus, IGF's are important mediators of β-cell replication in fetal and neonatal rat islets, but not in postnatal development (Billestrup N, Martin J.
M, Endocrinol. , 116: 1175-81, 1985; Rabin.
ovitch A, et al. , Diabetes, 32: 307-12,1.
983; Swenne I, Hill DJ, Diabetologia 32.
: 191-97, 1989; Swene I, Endocrinology, 1
22: 214-218, 19, 1988; Whittaker PG, et a.
1, Diabetologia, 18: 323-328, 1980). further,
Platelet-derived growth factor (PDGF) also stimulates fetal islet cell replication and its action does not require increased production of IGF-I (Swenne I, Endoc.
rinology, 122: 214-218, 1988). Furthermore, the effect of growth hormone on rat fetal β-cell replication appears to be largely independent of IGF-I (Romanus JA et al., Diabetes 34:
696-792, 1985; Swenne I, Hill DJ, Diabet.
32: 191-197, 1989). In the adult pancreas, I
GF-I mRNA is localized in D-cells. IGF-I is also found in the plasma membrane of β- and A-cells and scattered duct cells, but not in acinar or vascular endothelial cells (Hansson).
HA et al. , Acta Physiol. Scand. 132: 5
69-576, 1988; Hansson HA et al. , Cell
Tissue Res. 255: 467-474, 1989). This is in contrast to one report (Smith F et al, Diabetes.
, 39 (suppl 1); 66A, 1990), IGF-I expression in the tubule (du).
ct) and vascular endothelial cells and appeared to regenerate in endocrine cells after partial pancreatectomy. IGF's have not been shown to stimulate adult ductal cell or islet growth. IGF's also do not stimulate exocrine pancreas growth (Mossner J et al., Gut 28: 51-.
55, 1987). Thus, it is clear that the role of IGF-I, especially in the adult pancreas, is by no means certain.

Fibroblast growth factor (FGF) was found to initiate trans-differentiation of retinal pigment epithelium to intermediate retinal tissues in chicken embryos in vivo and in vitro (Hyuga M et al., Int. J. Dev. Biol.
993; 37: 319-326; Park CM et al. , Dev. Bi
ol. 1991; 148: 322-333; Pittack C et al.
, Development 1991; 113: 577-588). Growth factor β
Transforming (TGF-β) has been demonstrated to induce transdifferentiation of mouse mammary epithelial cells into fibroblasts [20]. Similarly, epidermal growth factor (
EGF) and cholera toxin were used to enhance ductal epithelial cyst formation among acinar cell fragments of the pancreas (Yuan S et al., In vitro Cell D).
ev. Biol. , 1995; 31: 77-80).

Searches for factors that mediate cell differentiation and survival must include both the cell and its microenvironment (Bissell MJ et al., JT.
heor. Biol. , 1982; 99:31), because the behavior of cells is controlled by other cells as well as the extracellular matrix (ECM) (Stocker).
AW et al. Curr. Opin. Cell. Biol. , 1990; 2
: 864). The ECM is a functional complex of molecules that functions as a scaffold for soft tissue cells and non-soft tissue cells. Its importance in pancreatic development is emphasized by the role of fetal mesenchyme in cell differentiation of epithelial cells (Bencosme SA
, Am. J. Pathol. 1955; 31: 1149; Gepts W, de.
Mey J. Diabetes 1978; 27 (suppl.1): 251.
Gepts W, Lacompte PM. Am. J. Med. , 1981;
70: 105; Gepts W .; Diabetes 1965; 14: 619;
Gitens S. In: Go VLW, et al. (Eds) The E
ndocrine Pancreas: Biology, Pathobiolo
gy and Disease. (New York: Raven Press
, 1986) p. 21). ECM is found in two forms of stromal matrix and basement membrane (BM). BM is a macromolecular complex of another glycoprotein, collagen, and proteoglycans. In the pancreas, BM contains laminin, fibronectin, collagen types IV and V, and heparan proteoglycans sulfate (Ingber D. In: Go VLW, et al (e.
ds) The Pancreas: Biology, Pathology
and Disease. (New York: Raven Press, 1
993) p. 369). The specific role of these molecules in the pancreas has not yet been determined.

ECM has a profound effect on differentiation. Mature epithelium, which normally never expresses mesenchymal genes, can be induced to do so by suspension in collagen gel in vitro (Hay ED. Curr. Opin. In Cell. Bio.
l. 1993; 5: 1029). For example, mammary epithelial cells become dull and lose their differentiated phenotype when attached to plastic dishes or adherent collagen gels (Emerman JT, Pitelka DR. In v.
intro 1977; 13: 316). When the gel is allowed to shrink, the same cells spin, polarize, secrete milk proteins, and accumulate continuous BM (
Emerman JT, Pitelka DR. In vitro, 1977;
13: 316). That is, different degrees of BM persistence or remodeling are critical to cell survival and maintenance of a normal epithelial phenotype (Hay ED. Curr. Opin.i.
n Cell. Biol. 1993; 5: 1029).

During tissue growth and in the presence of appropriate growth factors, cells are temporarily relieved of ECM-determined survival stress. It is now clear that there are two components of the cellular response to ECM interactions, one being the physical component, which involves morphological changes and cytoskeletal organization; the other is the biochemical component. It involves integrin clustering and increased phosphorylation of protein tyrosine (Ingber
DE, Proc. Natl. Acad. Sci. USA, 1990; 87: 35.
79; Roskelley CD et al. , Proc. Natl. Aca
d. Sci. USA, 1994; 91: 12378).

In addition to its known regulatory role in cell growth and differentiation, ECM has only recently been recognized as a regulator of cell viability (Bates RC, Lincz.
LF, Burns GF, Cancer and Metastasis R
ev. , 1995; 14: 191). The disruption of cell matrix ties is associated with apoptosis (Frisch SM, Frances H. J. Cell. Bio).
l. , 1994; 124: 619; Schwartz SM, Bennett.
MR, Am. J. Path. , 1995; 147: 229), which guides a series of morphological events (Kerr JFK et al., Br. J. Cancer, 19).
72; 26: 239), suggesting a process of active cell self-destruction.

According to one aspect of the present invention, the underlying technique is based on a combination of the above observations and is based on a combination of the above observations for the preparation of dedifferentiated intermediate cells from the islets of adult pancreatic islets of Langerhans. Incorporate enough of the following ingredients: 1. A solid matrix that allows "three-dimensional"culture; 2. The presence of matrix proteins including, but not limited to, collagen type I and laminin; C, including but not limited to growth factor EGF and cholera toxin and forskolin
AMP promoter.

A preferred feeding medium is DMEM / F12 supplemented with 10% fetal bovine serum. In addition, the starting tissue must be freshly isolated and cultured without pure purification.

The use of solid gels containing matrix proteins is an important part of the culture system, since extracellular matrix may drive the process of transdifferentiation. This point is highlighted by isolated pancreatic acinar cells and transdifferentiates into a tube-like structure when trapped in Matrigel basement membrane (Arias AE).
, Bendayan M, Lab Invest. , 1993; 69: 518-.
530) or highlighted by colored epithelial cells of the retina and transdifferentiated into neurons when plated on laminin-containing substrates (Reh TA et.
al. , Nature 1987; 330: 68-71). Most recently Gi
ttes et al. demonstrated that a defective pathway for epithelial growth of the embryonic pancreas formed islets using embryonic mouse pancreas at day 11 (Gitetes GK et.
al. , Development 1996; 122: 439-447). In the presence of basement membrane structures, however, the pancreatic rudimentary epithelium appears to be programmed to form ducts. This finding once again emphasizes the interrelationship between the duct and the islet,
And it emphasizes the important role of extracellular matrix.

This completes Stage 1 of the above process (production of dedifferentiated intermediate cells). During the first 96 hours of culture, the islets lost the progression of insulin gene expression (2
) Loss of immunoreactivity for insulin protein, and (3) cyst transformation associated with the emergence of CKA19, a marker of ductal cells (Arias AE,
Bendayan M, Lab. Invest. , 1993; 69: 518-5.
30). After the transformation is complete, the cells have the ultrastructural appearance of primitive tube-like cells. The cystic enlargement after the first 96 hours is, at least in part, associated with a profound increase in cell replication. These findings are consistent with the transdifferentiation of pancreatic islet cells into ductal cells (Yuan et al., Differentiation, 1
996; 61: 67-75, showing that isolated islets embedded in collagen type I gel in the presence of defined medium undergo cyst transformation within 96 hours).

Step 2-Production of functional β-cells requires a complete change in culture conditions. The cells are removed from the digested matrix and placed in basal liquid medium such as CMRL 1066 supplemented with 10% fetal bovine serum, including but not limited to fibronectin (10-20 ng / ml), IGF-1 (100 ng / ml). ), IGF-2
(1 oong), insulin (10-100 μg / ml), NGF (10-10)
Suspension with addition of soluble matrix protein containing 0 ng / ml) and growth factors. In addition, the glucose concentration should be increased above 11 mM. The additional culture adducts may include specific inhibitors of known intracellular signaling pathways of apoptosis, including but not limited to p38 specific inhibitors.

Evidence of reversion to the islet cell phenotype is (1) reappearance of the solid body sphere structure; (2) CK
-19 loss of expression; (3) evidence of endocrine granules on electron microscopy; (4) re-emergence of proinsulin mRNA on in situ hybridization; (5) reversion of basal insulin release into the culture medium.

The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention rather than limit the scope of the invention. Example I Preparation of Basal Feeding Medium The purpose of this study was to elucidate the mechanisms involved in the process of transdifferentiation.

Canine pancreatic islets were isolated using Canine Liberase® and Euroficol at the Cobe 2991 Cell Separator.
Purified on 1 gradient. 12 freshly purified islets in collagen type I gel
Implant up to 0 hours, and (i) DMEM / F12 plus cholera toxin (CT)
(Ii) CT-loaded CMRL 1066; (iii) Forskolin-loaded CMRL 1066; and (iv) CMRL 1066 alone. At the 16th hour, the intracellular level of cAMP (fmol / 10 ³ pancreatic islets) was EL
Increased in groups (i)-(iii) (642 ± 17, 33) compared to group IV (106 ± 19, p <0.01) as measured by ISA.
8 ± 48, 1128 ± 221). Total intracellular cAMP (integrated area under the curve) at 120 hours was measured by routine histology for cytokeratin AE1 / AE3, immunocytochemistry, and loss of proinsulin gene expression on in situ hybridization, Consistent with the% of islets undergoing transdifferentiation (63
± 2, 48 ± 2, 35 ± 3, 8 ± 1).

To assess the role of matrix proteins and the three-dimensional environment, islets were embedded in collagen type I, Matrigel® and agarose gels and cultured in DMEM / F12 plus CT. Collagen type I and Matr
Islets in igel® demonstrated a higher rate of cystic transformation compared to agarose (0 ± 0%, p <0.001) (63 ± 2% and 71 ±, respectively).
4%). Furthermore, transdifferentiation of islet cells was partially blocked by pre-incubation of newly isolated islets with RGD motif-presenting synthetic peptides.

In conclusion, these studies confirm the possibility that newly isolated islets undergo trans-differentiation of epithelial cells. Estimated levels of intracellular cAMP and matrix proteins displayed in the three-dimensional structure require this transformation to be induced. The net nature of the resulting epithelial cells, and the reversibility of the process remains undetermined.

Example II Factors Modulating the Transformation of Langerhans Islets to Duct Epithelial-Like Structures Regarding animal protection guidelines under general anesthesia from the pancreas from 6 mixed-breed dogs (25-30 kg body weight) of both genders. Partially excised according to the Canadian Conference (Wang
RN, Rosenberg L (1999) J Endocrinology
163 181-190). Prior to resection, cannulate the duct of the pancreas to remove Li
Intravenous infusion with Berase CI ™ (1.25 mg / ml) (Boehringer Mannheim, Indianapolis, Ind., USA) was performed according to established protocols (Horaguechi A, Merrell RC (1981) Dia.
betes 30 455-461; Ricordi C (1992) Panc.
realistic islet cell transfer. pp
99-112. Ed Ricordi C.I. Austin: R.A. G. Lande
s Co. ) Made possible. COBE 2991 Cell Processo
3-step EuroFi using r (COBE BCT, Denver, CO., USA)
Purification was achieved by density gradient separation in a coll gradient (London NJ
M et al. (1992) Pancreatic islet cell
transplantation 113-123. Ed Ricordi C
． Austin: R.A. G. Landes Co. ). Final preparation is 50 to 50
It consisted of 95% dithizone positive structures with diameters in the range of 0 μm.

Experimental Design To assess the role of intracellular cAMP, freshly isolated islets were embedded in collagen type I gels (Wang RN, Rosenberg L (199).
9) J Endocrinology 163 181-190), and (i
) 10% FBS, EGF (100 ng / ml) (Sigma, St. Louis, Lewis, MO, USA) and cholera toxin (100 ng / ml) (Sigma, St. Louis,
MO, USA) with DMEM / F12 (GIBCO, Burlington, ON,
Canada); (ii) 10% FBS and cholera toxin (100 ng / ml) and 16
． CMRL1066 (GIBCO) supplemented with 5 M D-glucose; (iii
) CMRL 1066 with 10% FBS and 2 μM forskolin (Sigma, St. Louis, MO, USA); and (iv) CMR with 10% FBS.
Cultured in L1066. About 3000 islets per group per time point were used. The islets were cultured at 37 ° C in 95% air / 5% CO ₂ and the medium was changed every other day. Representative islets from each group were examined after isolation (0
Hours) and then at 1, 16, 36, 72 and 120 hours were examined using the following study.

The role of cell-matrix interactions in the process of cyst transformation was assessed by conducting the following series of experiments. First, to determine whether the above process requires a solid gel environment, the islets are treated with 10% FBS plus CT.
And suspension culture was performed in DMEM / F12 to which EGF was added. To determine whether the solid body gel environment and extracellular matrix proteins were independent requirements, islets were embedded in 1.5% agarose gel and 10% FBS plus CT and E
Maintained in DMEM / F12 with GF. Alternatively, islets of DME with 10% FBS plus CT and EGF added in the presence of soluble laminin (50 μg / ml) or fibronectin (50 μg / ml) (Peninsula Laboratories).
Suspension culture was performed in M / F12. To determine if the process was at least partially integrin mediated, the islets were incubated at 37 ° C for 60 minutes with RGD-.
Motif-containing GRGDSP peptide or control peptide GRGESP (400 μg
/ Ml) (Peninsula Laboratories). Finally, islets were also embedded in Matrigel ™ (Peninsula Laboratories, Belmont, Calif., USA) to determine if cyst transformation was dependent only on type 1 collagen. Morphological Analysis Immunocytochemistry Tissues were fixed in 4% paraformaldehyde (PFA) and embedded in 2% agarose according to standard protocols for dehydration and paraffin embedding (Wang.
RN, Rosenberg L (1999) J Endocrinology
163 181-190). One set of 6 serial sections (thickness 4 μm) was cut from each paraffin block.

Serial sections were processed for routine histology and pancreatic hormones (insulin, glucagon and somatostatin, Biogenex, San Diego, CA.,
USA) and whole-cytokeratin cocktail AE1 / AE3 (Dako, Carpinteria, CA., USA) using the AB complex method (streptavidin-biotin horseradish peroxidase; Dako). Immunostained as follows (Wang RN et al. (1994) Diabet.
LOGO 37 37 1088-1096). For cytokeratin AE1 / AE3, sections were pretreated with 0.1% trypsin. Sections were incubated overnight at 4 ° C with the appropriate primary antibody. Negative controls included omission of primary antibody.

In Situ Hybridization Human Proinsulin (Novocastra, Burlington, ON, Canada)
In situ hybridization was performed at 120 hours on serial sections of freshly isolated islets and on epithelial cyst structures. The sections were hybridized with the fluorescein-labeled oligonucleotide cocktail solution for 2 hours at 37 ° C. Rabbit F with slides then combined with alkaline phosphatase
Incubated with ab anti-FITC antibody (diluted 1: 200) for 30 minutes at room temperature. The reaction product was converted to nitroblue tetrazolium and 5'-bromo-4-
Visualization was performed by a kit of enzyme-catalyzed color reaction using chloro-3-indolyl-phosphonate (Wang RN, Rosenberg L (1999) JE.
ndocrinology 163 181-190, Wang RN et.
al. (1994) Diabetologia 37 1088-1096).

Analysis of intracellular cAMP levels Cells were harvested from collagen gel and washed in cold 1 mM PBS. Two
After addition of 00 μl lysis buffer, each sample was sonicated for 30 seconds and then incubated for 5 minutes at room temperature. 100 μl of cell lysate was transferred to a donkey anti-rabbit Ig coated sample. Intracellular cA of non-acetylated sample
MP content was measured using a commercially available cAMP enzyme-linked immunoassay kit (
Assay range 12.5-3200 fmol / well, Amersham, Little Chalfont, UK). Data are expressed as fmol per 10 ³ islets. Insulin content assay Insulin content of cells is measured by solid phase radioimmunoassay (Immunocorp,
Montreal, Quebec, Canada) (Wang RN, Rosenberg
L (1999) J Endocrinology 163 181-190) was used to measure sensitivity of 26.7 pmol / l (0.15 ng / ml), assay intervariability of <5%, and 100% accuracy. The kit uses an anti-human antibody that cross-reacts with canine insulin. The values obtained were corrected for changes in cell number by measuring the DNA content using a fluorescent DNA assay (Y.
uan S et al. (1996) Differentiation 61
, 67-75). Data are expressed as μg per μg DNA. Cell death and proliferation Cells cultured in DMEM / F12-CT and CMRL1066 were recovered from the gel with collagenase XI (0.25 mg / ml) (Sigma, Montreal, Quebec) and cells in the process of apoptosis. Of a specially programmed cell death detecting histone-binding DNA fragments in the cytoplasmic demonstration of Escherichia coli
Processed for ISA (Roche Molecular, Montreal,
Quebec) (Paraskevas S et al. (2000) Ann.S.
during printing). Cells were incubated in lysis buffer for 30 minutes and supernatant containing cytoplasmic oligonucleosomes was measured at 405 nm absorption. Changes in sample size were corrected by measuring the total sample DNA content (Yuan S et al. (1996) Differentiatioio.
n 61,67-75).

To assess cell proliferation, cells cultured in DMEM / F12-CT and CMRL1066 were treated with 10 μM 5-bromo-2′-deoxyuridine (BrdU).
, Sigma) for 1 hour at 37 ° C. Harvested cells were fixed in 4% PFA as described above. Immunostaining for BrdU was performed using the AB complex method. Sections were pretreated with 0.1% trypsin and 2N HCl denatured DNA. Monoclonal anti-BrdU antibody was used at a dilution of 1: 500 (Sigma). To assess the BrdU labeling index, the number of cells positive for BrdU reaction was counted and expressed as a percentage of the total cell number counted. At least 500 cells were counted per section for each experimental group and time point.

Statistical Analysis Data obtained from 6 different islet isolates are expressed as mean ± SEM. Differences between groups were assessed by one-way analysis of variance. Results Morphological changes Under the inverted microscope, the newly isolated islets appeared as solid spheres. At this point, no cytokeratin-positive cells were demonstrated (Fig. 8A-B).

DMEM / F12 plus CT, CMRL1 embedded in type I collagen
For islets cultured in 066 plus CT or CMRL1066 plus forskolin, differentiation of ductal epithelium was first observed at about 16 hours, concomitant with loss of cells in the islet epithelium. At this point, the cells lining the cyst space were cytokeratin positive (FIGS. 8C-D). The fully developed structure was present for 72 hours in culture (Fig. 8E-F). Islets cultured in CMRL1066 alone maintained the appearance of solid spheres for the duration of the study and did not undergo epithelial transformation. Immunocytochemistry staining did not demonstrate co-localization of cytokeratin and islet cell hormones. This is consistent with cytokeratin staining observed only in ductal epithelial cells by observation in the complete pancreas. Proinsulin gene expression and insulin protein were progressively lost during tube epithelial differentiation (Fig. 9).

1 hour after intracellular cAMP, DMEM / F12-CT, CMRL1066-CT and CMRL
Intracellular levels of cAMP in islets maintained in 1066-Forskolin were significantly elevated compared to freshly isolated islets or islets maintained in CMRL 1066 alone (FIG. 10A). In fact, intracellular levels of cAMP in islets cultured in CMRL 1066 alone did not increase during the entire study period. Total intracellular cAMP (integrated area under the curve) measured over 120 hours was similar for islets cultured in DMEM / F12-CT, CMRL1066-CT and CMRL1066-Forskolin (respectively). , 15 ± 3
, 16 ± 2, 17 ± 3), the most sustained increase in cAMP was DMEM / F1.
2-CT in islets and exposed to both EGF and CT. In contrast, C
Islets cultured in MRL 1066 alone had the lowest total intracellular cAMP levels (4 ± 1, p <0.001) (FIG. 10B), which translates to the lowest levels of islet-tube transformation. (FIG. 10C).

Intracellular Insulin Content The cellular content of insulin (FIG. 11) was highest in newly isolated islets (11 ± 2 μg / μg DNA). After 16 hours of culture, DMEM / F
The insulin content of cells cultured in 12-CT, CMRL1066-CT and CMRL1066-forskolin dropped dramatically, by 120 hours to 7% of the initial value. Islets cultured in CMRL1066 alone did not undergo epithelial transformation and retained high levels of intracellular insulin compared to the other three groups (p <0.03, Figure 11).

Cell Death and Proliferation Analysis To determine whether the loss of cells during cyst transformation was due at least in part to programmed cell death, we used a special cell death ELISA. . At 16 hours, cytoplasmic oligonucleosome enrichment was significantly higher in islets cultured in DMEM / F12-CT compared to islets cultured in CMRL1066 alone (p <0.02. , FIG. 12A). After 36 hours, there were no differences between the groups. Looking at the data as a whole (FIG. 12A), a wave of apoptosis occurred in both groups of islets, but the time course of cell death was DMEM / F1.
It seems that the pancreatic islets undergoing cyst transformation within 2-CT are shifted to the left.

To assess proliferation, cells were labeled with BdrU. After isolation, DMEM / F1
The labeling index of BrdU cells in islets cultured in 2-CT was 0.8%, which was identical to that of islets cultured in -CMRL1066 alone. Three
After 6 hours, however, a wave of cell proliferation occurred later in the DMEM / F12-CT group and the labeling index reached 18% at 120 hours (Fig. 12B). By comparison, the labeling index for islets in CMRL 1066 remained essentially unchanged throughout the study (p <0.01).
. Role of the integrin-ECM interaction To determine whether elevated intracellular cAMP was sufficient to induce ductal epithelial differentiation, the islets were maintained in suspension culture in DMEM / F12-CT, and Not embedded in collagen gel. Under these conditions, epithelial transformation did not occur. This suggested that an increase in intracellular cAMP was necessary but not sufficient for transformation, and that matrix should also play an important role in the process.

To determine whether a solid gel environment or the presence of extracellular matrix proteins alone was required, the islets were agarose gel, type 1 collagen gel or Mat.
embedded in rigel ™. Only the islets embedded in the latter two gels underwent cyst transformation (Table 1). Furthermore, islets maintained in suspension in DMEM / F12-CT supplemented with either soluble laminin or fibronectin failed to undergo tube transformation. These experiments showed that the process of transformation required the presence of ECM proteins that were present in the solid gel environment.

[0068]

Table 1 Effect of extracellular matrix on islet-cyst transformation in isolated dog islets Time Matrigel Collagen agarose soluble laminin / I fibronectin ^a 16 hours 19 ± 4.7 14 ± 1.4 ― ― 36 hours 49 ± 3.7 35 ± 3.9--72 hours 60 ± 3.7 42 ± 1.6--120 hours 71 ± 4.5 63 ± 2.4--Transformation in a more direct manner To examine the role of integrin-mediated signaling in the process, islets were pre-incubated with the RGD motif-containing GRGDSP peptide prior to implantation in collagen. This resulted in 57% (p <0.002) of control DMEM / F12-CT group cyst transformation at 72 hours.
1) (Fig. 14A). The control peptide GRGESP had little effect on the transformation process. Pretreated islets with either soluble fibronectin or laminin prior to implantation were up to 50% of controls at 72 hours (p <0.
01) reduced cyst transformation (Fig. 14B). When the islets were pre-incubated with both GRGDSP and laminin, cyst transformation was 33% more than control.
(P <0.001, FIG. 14C). Discussion Differentiated cells normally maintain their cell specificity in adults, where the stability of the cell phenotype is related to the cell's interaction with its microenvironment. Perturbation or loss of stabilizing factors, however, may induce cells to change their commitment (Okada TS (1986) Development Growth Dif.
f 28,213-221). We have previously reported that isolated islets of Langerhans embedded in type 1 collagen gel can be induced to trans-differentiate into tube-like epithelial structures (Yuan S et al. (1996) Diff.
erentiation 61, 67-75).

Little is currently known about the molecular events involved in transdifferentiation.
Therefore, the purpose of this study was to characterize the factors involved in this transformation process in order to better understand the functional relationships that confer morphogenic stability to cells in isolated islets. Met. Given the poorer long-term success rate of cell-based therapies for diabetes mellitus, especially islet transplantation (Rosenberg L. (1
998) Int'l J Pancreatology 24, 145-168.
), Studies such as those described herein can provide new insights into the issues surrounding the issue of graft failure.

There are two important findings. First, the process of cyst transformation is intracellular c
We have demonstrated that it requires both elevated AMP and the presence of ECM proteins present as a solid support. Second, we have determined that the formation of cystic structures from solid islet spheres is a two-step process involving a wave of endocrine cell apoptosis followed by cell proliferation of novel duct-like cells. .

Detailed information is not available as signal transduction during transdifferentiation has been the subject of much recent research. However, information flow mediated by cAMP seems to play an important role (Ghee M, Baker H, et a.
l. (1998) Mole Brain Res 55, 101-114; Os.
aka H, Sabban EL (1997) Mole Brain Res
49, 222-228; Yarwood SJ et al. (1998) Mo
le Cell Endocrinol 138, 41-50). In this study, we found that elevated intracellular cAMP was necessary but not sufficient for induction of islet-to-cyst transformation. However, it was not simply the peak value of the increase in critical intracellular cAMP, but rather the persistence of the increase associated with the most frequent ductal epithelial transformation. Increased levels of cAMP produced less than the maximal transformation response, as produced by media supplemented with EGF alone or forskolin alone. The longest duration of cAMP was obtained in medium supplemented with the combination of EGF and CT. This is Yao
(Yao H, Labudda K, Rim C, et al. (1995).
J Biol Chem 270, 20748-20753), but they demonstrated a requirement for retained versus transient signaling in cAMP-mediated EGF-induced differentiation in PC12 cells. This discovery
It also serves the purpose of highlighting the similarities between pancreatic β-cells and cells of neuronal origin (Scharfmann R, Czernichow P (1997) Panc.
realistic grow and regeneration. pp170
-182. Ed Sarventnick N.M. Austin: Karger
Landes). Thus, as in other systems (Yao H, Labbudda
K, Rim C, et al. (1995) J Biol Chem 270
, 20748-20753), the cellular response of islet cells to the action of growth factors is
It may depend not only on activation of growth factor responses by specific growth factors, but also on synchronous signaling that enhances intracellular signaling such as cAMP.

The increase in intracellular cAMP is also of interest because elevated cAMP may form part of the effector system that controls apoptosis in pancreatic β-cells (Loweth AC, Williams GT, et al. (19
9) FEBS Lett 400, 285-288). Thus, it should be noted that cell loss by apoptosis is the first step we observed in the process of islet to cyst transformation. Although the apoptosis should occur during islet transformation in this system of interest, the islets are embedded in collagen gels and such a matrix differentiates different types of cells in culture. It has been reported to help promote or maintain the condition (Foster CS et al. (1983) Dev Biol 9).
6, 197-216; Yang J et al. (1982) Cell Bi
ol Int 6,969-975; Rubin K et al. (1981
) Cell 24,463-470). On the other hand, extracellular matrix may also enhance the process of transdifferentiation. This is due to the isolated pancreatic acinar trans-differentiating into a tube-like structure when trapped in Matrigel ™ (Arias AE, Bendayan M (1993) Lab Inve.
st 69, 518-530) and by retinal pigment epithelial cells that transdifferentiate into neurons when placed on a laminin-containing substrate (Reh TA et al.
． (1987). Nature 330, 68-71), emphasized. Most recently, Gitetes et al. (Gitetes GK et al. (1996) Dev.
elopment 122, 439-447) demonstrated that a defective pathway for the growth of embryonic pancreatic epithelium forms pancreatic islets using day 11 embryonic mouse pancreas. In the presence of basement membrane structures, however, the pancreatic rudimentary epithelium appears to be programmed to form ducts. This finding again highlights the interrelationship between ducts and pancreatic islets and the important role of the extracellular matrix. Despite these observations, the presence of solid phase ECM support appears to be necessary, but the first step is a sufficient condition for transformation of solid islets into cystic epithelial-like structures involving apoptotic cell death. is not.

The conversion of solid bodies into hollow structures is a morphological process that is frequently observed during vertebrate embryogenesis (Coucouvanis E, Martin GR (1
995) Cell 83, 279-287). In the early mouse embryo, this process of cavity formation transforms the ectoderm of the embryo into the columnar epithelium surrounding the cavity. Cavity formation has been proposed to be the result of the interaction of two signals, one of ectodermal cells that acts for a short time to create a cavity by inducing apoptosis of the inner ectoderm cells. From the outer layer, and the other is a rescue signal mediated by contact with the basement membrane, which is required for the survival of columnar cells (Coucouvani).
s E, Martin GR (1995) Cell 83, 279-287).
The combination of these two signals results in the death of inner cells not in contact with the ECM and in the survival of outer cell monolayers in contact with the basement membrane. A central feature of this model is the direct initiation of apoptosis by external signals that cause cell death. The second key feature of the above model is a signal that seems to be mediated by attachment to the ECM and rescues cells from cell death. Ultimately, there are a wealth of examples of cell dependence on ECM for survival (Meredith).
JE et al. (1993) Mol Biol Cell 4,953-
961; Boudreau N, Sympson CJ, et al. (199
5) Science 267, 891-893). In our model of islet-cyst transformation, external death signals are probably provided by those factors that increase intracellular cAMP. Moreover, the observation that cell loss occurs preferentially in the center of islets during transformation, supports the notion that the ECM acts as a rescue signal for those cells in the periphery. The exact role of integrins in this process has yet to be described more completely. Integrin-ligand binding does not require itself to contribute to survival signals. For example, integrins can modulate cell responsiveness to growth factors (E
lliot B et al. (1992) J Cell Physiol 1
52, 292-301).

One area that was not explored in this study was the reversibility of the transformation process. Reversibility of trans-differentiation was reported in other cell lines (Eren
preisa J, Roach HI (1996) Mechanisms of
Aging & Development 287, 165-182). Transdifferentiation may involve cell proliferation and the appearance of pluripotent dedifferentiated intermediate cells (Y
uan S et al. (1996) Differentiation 61
, 67-75), capable of expressing markers characteristic of several alternative phenotypes. This is true in our system (Yuan S et al. (1996)
) Differentiation 61, 67-75). That is, it may be possible to grow a population of pluripotent cells and then induce the induced differentiation to the desired cell phenotype—in this case mature insulin-producing β-cells. The in vitro system used in these studies was unique for two reasons-it required fetal tissue, and the starting tissue, adult islets, was well defined.

In summary, this study extends our previous observation that adult islets of Langerhans can be transformed into ductal epithelial cystic structures by a two-step process involving apoptosis and subsequent cell differentiation and proliferation. The precise biochemical mechanism appears to include, at least in part, the elevation of intracellular cAMP mediated by the combination of cholera toxin and EGF, and the survival signal contributed by the solid phase ECM support. The differentiation potential of cells containing novel epithelial structures remains to be completely elucidated.

Although the present invention has been described in relation to particular embodiments thereof, further modifications are possible, and as applicable to known or customary experience in the field to which the invention pertains, and Any modifications, uses, or adaptations of the invention are generally followed in accordance with the principles of the invention, including deviations from the present disclosure, as may be applied to essential features of the invention, and as subject to the scope of the claims. It is to be understood that this application is intended to do so.

[Brief description of drawings]

FIG. 1 shows the conversion of cell types from islets to a duct-like structure (human tissue), (a) islets in the pancreas, (b) islets after isolation and purification, (c). ) Shows progressive formation of cystic structures with complete loss of islet morphology, (df) islets in the solid phase beginning to undergo cystic changes.

FIG. 2 shows the same progression of the changes shown in FIG. Cells are stained for insulin by immunochemistry. (A) Islet in the pancreas. (B) Islet after isolation and purification. (C
-E) Progressive loss of islet phenotype. (F) High-power view of the cyst wall consisting of tube-like epithelial cells. One cell still contains insulin (arrow).

FIG. 3 shows the same progression of the changes shown in FIG. Cells are stained for glucagon by immunocytochemistry. (A) Islet in the pancreas. (B) Islet after isolation and purification.
(Ce) Progressive loss of islet phenotype. (F) High-power view of the cyst wall consisting of tube-like epithelial cells. One cell still contains glucagon (arrow).

FIG. 4A-C shows evidence of cell phenotype by CK-19 immunocytochemistry. Upper left panel-cystic structure in solid phase matrix. All cells stained for CK-19, marker expressed in ductal epithelial cells in pancreas. Bottom panel-suspension culture. A structure that presents both epithelial-like and solid body components. Upper right panel-C only epithelial-like component
Retains K-19 immunoreactivity. The solid body component has lost its CK-19 expression and appears to be islet-like.

5A-B: Upper panel-ultrastructural appearance of cells composed of cystic structures in a solid phase matrix. Note the loss of fine villi and endocrine granules. Bottom panel-ultrastructural appearance of cystic structures removed from solid phase matrix and placed in suspension culture. Note the reduction of fine villi and the reappearance of endocrine granules.

6A-B show in situ hybridization of proinsulin mRNA. Upper panel-cystic structure with virtually no cells containing message. Bottom panel-cystic structures were removed from the matrix and placed in suspension culture. Note the current appearance of both solid bodies and cystic structures. The solid structure is proinsulin mRNA
With abundant expression of.

FIG. 7 shows insulin release into the culture medium according to the structure observed in the bottom panel of FIG. Note that there is undeniably no insulin secreted from tissues when in the cystic state (far left column). FN-fibronectin;
IGF-1-insulin-like growth factor-1; Gluc-glucose.

FIG. 8 shows DMEM / F12-CT embedded in a collagen matrix.
Shown islets retained therein. Pictures under an inverted microscope (A, C, E) and corresponding tissue sections stained for pancytokeratin AE1 / AE3 by immunocytochemistry (B, D, F). (A, C, E, x100; B, D, F, x200)

FIG. 9 shows islets in the intermediate state of cyst transformation that still contain (A) cells expressing proinsulin mRNA and (B) cells that synthesize and store insulin protein. (X400)

FIG. 10A shows intracellular levels of cAMP during the time course of pancreatic islet-cyst transformation. Note the relatively constant level of intracellular cAMP in islets retained only by CMRL 1066. FIG. 10B shows the integrated amount of cAMP (area under the curve of A) measured at 120 hours. There were no differences observed between islets cultured in DMEM / F12-CT, CMRL-CT and CMRL-forskolin. However, C
Note that islets retained only in MRL had significantly lower intracellular cAMP. FIG. 10C shows the percentage of islets that underwent increased cystic transformation over time during the culture period of DMEM / F12-CT, CMRL-CT and CMRL-forskolin groups. The islets retained in CMRL 1066 had very low levels of intracellular transformation and remained constant. * P <0.05, ** p <0
． 01, *** p <0.001.

FIG. 11 shows the progressive loss of tissue insulin content during the time course of cyst transformation. DMEM / F12, corresponding to the early onset of apoptosis by 16 hours
Note the steep drop in islets retained in -CT, CMRL-CT and CMRL-forskolin. * P <0.03.

FIG. 12: DMEM / F12-CT and CMRL during the time course of cyst transformation.
2 shows the apoptotic activity (A) and BrdU labeling index (B) of islets cultured in 1066. Note the shift to the left of the onset of islet apoptosis in DMEM / F12-CT. * P <0.02; *** p <0.01; *** p <0.00
1.

FIG. 13: Integrin binding peptides GRGDSP and GRGESP (A)
, Extracellular matrix proteins laminin and fibronectin (B), and GRG
Figure 3 shows the effect of DSP or GRGESP and laminin (C) on islet cyst transformation. * P <0.05, ** p <0.01. *** p <0.001.

FIG. 14 shows the effect of extracellular matrix on islet-cyst transformation of isolated canine islets.

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Claims (8)

[Claims] 1. A medium for preparing dedifferentiated cells derived from post-natal islets of Langerhans, which is in a physiologically acceptable culture medium in an effective amount: a) a solid phase for three-dimensional culture. A matrix environment; b) a soluble matrix protein; and c) a medium containing first and second factors for developing, retaining and growing the dedifferentiated intermediate cells. 2. The medium of claim 1, wherein the first factor induces an increase in intracellular cAMP and the second factor is derived from acinar cells. 3. The medium of claim 1, wherein the culture medium comprises DMEM / 12 supplemented with an effective amount of fetal bovine serum. 4. The medium of claim 1, wherein the first factor comprises one or more cholera toxin (CT), forskolin, high concentrations of glucose, a cAMP promoter, and EGF. 5. The medium of claim 1, wherein the matrix protein comprises one or more laminin, collagen type I and Matrigel ™. 6. A method for preparing dedifferentiated cells derived from postnatal Langerhans islet neurons, which comprises contacting the cells with the medium according to any one of claims 1 to 5. 7. An in vitro method for the expansion of stem cells, comprising the steps of: a) preparing stem cell-derived dedifferentiated intermediate cells; and b) dedifferentiating intermediate cells. A method comprising: growing in vitro in a medium according to any one of claims 1 to 3; and c) inducing stem cell differentiation characteristics of the expanded cells in step b) in vitro. 8. The method of claim 7, wherein the stem cells are expanded from the group consisting of muscle, skin, bone, cartilage, lung, liver, bone marrow and hematopoietic cells.