AU724745B2 - Methods for the isolation and proliferation of insulin-secreting cells - Google Patents

Description

WO 97/49728 PCT/US97/11018 Methods for the Isolation and Proliferation of Insulin-Secreting Cells Field of the Invention The present invention relates to methods and compositions useful for the proliferation of pancreatic islets of Langerhans as a therapy for diabetes mellitus, optionally including their delivery encapsulated within microcapsules. In particular, invention methods result in the expression of certain characteristic intracellular proteins HNF3 beta, NKX 6.1, STF-I, or beta2) whose activity regulates the transcription of the insulin gene.

Backgcrround of the Invention Insulin-dependent diabetes is characterized by the loss of insulin-producing beta cells from the pancreatic islets of Langerhans. Standard therapy has included parenteral administration of insulin (either bovine or porcine or recombinant human) by means of multiple daily injections or an indwelling catheter-andpump. This treatment, however, can only temporarily delay the pathological complications of the disease.

Adult human pancreatic islets have been transplanted into patients in efforts to achieve independence from insulin injections. This approach suffers from inadequate supplies of human islets and the complications of graft rejection. Immunosuppressive treatment with cyclosporine A) has been limited by the toxic side effects and by the increased potential for infection. These limitations have necessitated the search for an improved source of islet cells.

Fetal pancreatic islets contain many beta stem cells which can mature after transplantation and which are Ii WO 97/49728 PCTIUS97/11018 2 less subject to rejection by the recipient, but they cannot be obtained in large enough amounts to serve as a practical therapeutic approach. Transplantation of individual cells or cellular communities (including human ,or porcine pancreatic islets) will depend on an inexhaustible supply of functional living cells which can be used in experimental models as well as in human therapy.

The endocrine pancreas consists primarily of three cell types which are distinguished by their selective synthesis and secretion of the peptide hormones glucagon (from A-cells), insulin (from B-cells), and somatostatin (from D-cells). Embryologically derived from the small intestine, these pancreatic islet cells may retain regulatory pathways which originated in the gut to direct expression of the same peptide hormone genes. In its early stages, the fetal pancreas is populated by pluripotent stem cells which can co-express somatostatin, insulin, and glucagon. As these stem cells mature, their endocrine hormone repertoire becomes restricted to expression from a single gene instead of three genes simultaneously.

Although common transcription factors may initially regulate all three genes, distinct nuclear transcription factor proteins subsequently specify tissue-transcription factor proteins subsequently specify tissue-specific peptide production in individual mature islet cell types.

Expression of the somatostatin gene in rat pancreatic islet cell lines has been shown to require multiple copies of a tissue-specific promoter element (TSE) in close physical association with the somatostatin promoter. The somatostatin TSEs contain a structural feature which is generally recognized by homeobox-type proteins ]Leonard et al., MOLECULAR ENDOCRINOLOGY vol. 7:1275-1283 (1993)].

Because the promoter DNA regions required for islet expression of insulin, somatostatin, and glucagon include these critical AT-rich structural features, it has h, WO 97/49728 PCT/US97/11018 3 been presumed that homeobox-containing factors are important activators of these genes. The homeobox is a sequence specific DNA-binding motif present in numerous developmentally regulated transcription factors, some of which are thought to be required for the expression of lineage-specific genes [Guz, et al., Development vol.

121:11-18 (1995)].

A number of homeobox-type factors have been proposed as key regulators of individual genes in the pancreas, including the insulin gene. In particular, there is now compelling evidence that STF-l is required for pancreogenesis and constitutes a step in directing betacell lineage. Subsequently, STF-I binds to two of the insulin promoter-proximal TSEs and directs high-level expression of the insulin gene within insulin-producing cells in synergy with other binding factors, such as Beta2.

Furthermore, STF-l is not expressed in glucagon-producing cells, suggesting that the differential expression may be important in the divergence of these cell types during development [Peers, et al., MOLECULAR ENDOCRINOLOGY VOL.

8:1798:1806 (1994)].

Accordingly, there remains a need in the art for methods to generate an inexhaustible supply of functional living cells which can be used in human therapy, as well as in experimental models of diabetes mellitus.

Brief Description of the Invention In accordance with the present invention, it has been discovered that the overall rate of insulin secretion obtained from proliferated insulin-secreting mammalian cells can be partially determined by the activity of certain intracellular transcription factors HNF3beta, STF-l, NKX 6.1 or Beta2) which exert direct effects upon the insulin promoter and thereby alter its WO 97/49728 PCT/US97/11018 4 transcriptional activity. This effect is sufficiently pronounced in proliferated insulin-secreting cells that measurable amounts of these transcription factors can be detected using an appropriate assay.

These means for monitoring insulin production enable the development of methods for isolating insulinsecreting cells from mammalian islet cells, methods for proliferating insulin-secreting cells, and methods for optimizing the insulin-secreting ability of mammalian islet cells.

Detailed Description of the Invention In accordance with the present invention, there are provided methods for the isolation of insulin-secreting cells from mammalian islet cells, said methods comprising: culturing said islet cells under conditions which promote insulin secretion, and isolating those cells which produce one or more transcription factors associated with insulin production.

Culturing conditions suitable for use herein can be readily identified by those of skill in the art. For example, Ham's F12 medium (GibcoBRL #317865-035) containing in the range of about 2-4% fetal bovine serum and in the range of about 10-200 Ag/ml of bovine pituitary extract (Pel Freeze Biologicals #57133-2) can be employed at a temperature of about 37 0 C in an atmosphere containing about

CO

2 in 10 mm culture dishes. As readily recognized by those of skill in the art, a variety of other media are suitable for use herein, media containing 0-400 ng/ml of insulin, 0-100 ng/ml of placental lactogen, 0-10 micromolar hydrocortisone, 0-5 mM nicotinamide, 0-200 U/ml of hepatic growth factor, triiodothyronine, 1-5 yg/ml of apotransferrin, 20-500 Ag/ml of hypothalamic extract, 0-2% bovine serum albumin, and the like.

WO 97/49728 PCT/US97/11018 As used herein, the phrase "homeobox-type transcription factor" or "transcription factor protein" refers to a protein which is able to bind to native promoter regions of pancreatic islet hormone genes and thereby modulate mRNA transcription. As used herein, "homeobox" refers to a domain of about 55-65 amino acids within the transcription factor which binds to specified nucleotide sequences within the given gene promoter region.

The homeobox domain of the transcription factor preferably binds to either one or both of the "tissue-specific promoter element(s)" (TSEs) promixal to the insulin promoter. The transcription factors have the ability to transactivate the gene expression of pancreatic hormones.

The transcription factor STF-1 (also known as IDX-1, IPF-1, and PDX-1) accounts for the predominant TSE-binding activity in nuclear extracts from insulin- and somatostatin-producing pancreatic islet cells, supporting the proposition that this protein plays a primary role in regulating peptide hormone expression and in specifying endocrine cell lineage in the developing pancreas.

Transcription factors are uniformly expressed in B-cells and the D-cells of the endocrine pancreas, and are not expressed in exocrine cells. Those of skill in the art can readily identify a variety of transcription factors associated with insulin production, NKX 6.1, HNF3-beta, STF-1, Beta-2, and the like.

As used herein, the phrase uniformly expressed means that naturally occurring RNA encoding the transcription factor protein can be detected in each of the pancreatic islet cell types which produce insulin.

Methodologies such as immunohistochemical and molecular biological techniques involving antibodies against STF-1 make it possible to monitor and analyze STF-1 expression from transcription of DNA into mRNA to translation of the mature protein, and correlate this with WO 97/49728 PCT/US97/11018 6 insulin production. Thus, STF-1 can be used as an identifying marker to detect specific functional activity of the insulin promoter, concurrent with insulin expression in the beta cells of pancreatic islets. This STF-1 factor can be used as a probe, not only to optimize cell culture media in terms of developing a media which provides the highest STF-1 activity, but also provides a probe for identifying STF-1 cells, and thus insulin-producing cells.

STF-1 is a member of the homeobox class of transcription factors and is required for pancreatic organogenesis [Johnson, et al., NATURE 371: 606-609 (1994)]. It is expressed during early development by the epithelial cells of the gut and most of the cells that will eventually form the pancreas. However, in the adult, STF-1 expression is lost in pancreatic ductal, exocrine, and Acells, and is restricted to the duodenal epithelium, beta cells and a minor subset of delta cells [Gus, et al., DEVELOPMENT 121:11-18 (1995)].

In adult beta and delta cells, STF-1 is required for the hallmark phenotype of these cells, the expression of insulin in beta cells [Peers et al., MOLECULAR ENDOCRINOLOGY 7:1798-1806 (1994)] and somatostatin in delta cells [Leonard et al., MOLECULAR ENDOCRINOLOGY 7:1275-1283 (1993)]. STF-1 binds to the CT2 box in the human insulin promoter [Petersen et al., PROC. NAT. ACAD. SCI. USA 91:10465-10469 (1994)] resulting in increased transcription of the insulin gene.

According to the present invention, other transcription factors, STF-1, HNF3 beta, NKX 6.1, Beta2, and the like, can be utilized as both markers for mature islet cells and as a requirement for insulin expression. Further according to the present invention, it has been discovered that culture conditions can be optimized based upon the expression levels and activities WO 97/49728 PCT/US97/11018 7 of such transcription factors in conjunction with measurements of glucose-responsive insulin release.

In accordance with another aspect of the present invention, there are provided methods for proliferating insulin-secreting cells, said methods comprising: culturing mammalian islet cells under conditions which promote insulin secretion, isolating those cells which produce one or more transcription factors associated with insulin production, and culturing the isolated cells under conditions which promote production of one or more of said transcription factors.

In accordance with yet another aspect of the present invention, there are provided methods for optimizing the insulin-secreting ability of mammalian islet cells, srid methods comprising: culturing said islet cells under a variety of culturing conditions which promote insulin secretion, and selecting those conditions which stimulate production of elevated levels of one or more transcription factors associated with insulin production.

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

Example 1 Porcine and human islets are isolated by standard techniques. The isolated islets are cultured in standard culture vessels utilizing commercial cell culture media with the exception that, in contrast to standard cell culture where oxygen tension, glucose levels and pH fluctuate between high and very low levels, in accordance WO 97/49728 PCT/US97/11018 8 with the present invention, islet cells are proliferated at physiological levels of oxygen, glucose and pH. Islets proliferated in this manner can be defined by the STF-1, HNF3 beta, NKX 6.1, and Beta2 protein levels as determined by Western blot analysis.

For example, the presence of STF-1 protein can be used to characterize the proliferating cells. Cultures of proliferated islets (10 cm plates) at passage 1 and passage 4 are extracted at 65 0 C with 0.5 ml each Cell Lysis Solution sodium dodecyl sulfate, 1 mM EDTA, 1 mM 2mercaptoethanol, 100 mM KC1, 20 mM Tris HC1, pH 7.4).

Equivalent amounts of each extract (10 micrograms protein) are electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. The blot is probed with an STF-1 specific antibody [Peers et al., MOLECULAR ENDOCRINOLOGY 8:1798-1806 (1994)] (1:1000 in 1% gelatin, 0.02% Tween 20, 500 mM NaC1, 20 mM Tris-HCl pH 7.5) and detected with [I1 25 ]-protein A (Amersham IM 144, 1:1000 in mM Tris-HC1 pH 7.5, 500 mM NaC1, 0.5% bovine serum albumin, 0.5% Triton X-100, 0.2% SDS) followed by autoradiography.

Example 2 This example describes the quantitation of STF-1 mRNA levels for use as specific probes for beta cells. The proliferated beta cells from representative islet cultures are extracted with TriReagent (Sigma #T-9424) and both RNA and protein samples are prepared. The levels of STF-1 mRNA are determined by quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) utilizing primers that distinguish the correctly spliced message, e.g., 5'-GGACTCACTGTATTCCACTGGCATCAA-3' (SEQ ID NO:1); 5'-TGAAGTGGAAAAAGGAGGAGGACAAGA-3' (SEQ ID NO: 2).

WO 97/49728 PCT/US97/11018 9 Northern blot analyses of selected samples are used to confirm the RT-PCR results. High level STF-1 expression is seen in cultures containing predominantly pancreatic endocrine cells but not in cultures of acinar cells or fibroblasts. By comparing the expression of STF-1 with other markers insulin and somatostatin) the proliferative capacity of the beta cells in the culture is determined and optimized for each experiment. The culture conditions for each donor islet cell population is tailored based upon these results.

Example 3 This example demonstrates the use of the level and activity of the STF-1 protein to optimize beta cell culture medium. The interaction of STF-1 with the CT2 element in the insulin promoter is augmented in insulinoma cells following glucose stimulation implying that both the level and the state (phosphorylation, glycosylation etc.) of the STF-1 protein is important for insulin expression.

Islet cell culture conditions are optimized in part based on the expression level and glucose-dependent modification of the STF-1 protein. The levels of STF-1 protein are determined in the protein extracts by Western blot analyses using specific antibodies [Peers et al., MOLECULAR ENDOCRINOLOGY 8:1798-1806 (1994)] and compared with control protein and other markers.

Under optimal islet cell proliferation conditions, STF-1 protein levels normalized for cell number remain constant. The glucose-dependent modification of STF-1 is examined in cell extracts by electrophoretic mobility shift assays, DNA footprinting experiments, and by isoelectric focusing'. -The results of the assays are compared with measurement of glucose-responsive insulin release as determined with Static Glucose Stimulation assays and used to optimize the culture conditions.

WO 97/49728 PCT/US97/11018 This is the first report of the use of the state and level of STF-l protein to optimize beta cell culture conditions. In addition, islet cells have been identified in representative cultures by in situ hybridization with a STF-1 probe. Co-localization of STF-I and other beta-cell markers in the cultured cells allows the morphological identification of insulin-producing cells leading to the sub-culturing of homogeneous beta cell clones. Physical isolation of these clones may be possible by robotic instrumentation, including the use of laser directed splicing of the cell and retrieval of these clones from the cell culture vessel for further subculture. This technique of using STF-l as a probe has never been described to confirm specific beta cell selection and to optimize media for beta cell selection.

Example 4 This example describes means to regulate the STF-l promoter. High level expression of STF-I is believed to be required for the beta cell phenotype. In a transgenic mouse model, an STF-l promoter fragment has been shown to direct the expression of a beta-galactosidase reporter gene predominantly to the pancreatic beta cells.

It is possible to introduce a similar reporter construct into proliferating beta cell populations and monitor the effect of the media components in order to identify factors that maximize transcription from the STF-I promoter.

Reporter gene expression data can be correlated with other data and conditions optimized for glucose-dependent insulin release.

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

WO 97/49728 PCTIUS97I1 1018 11 SEQUENCE LISTING SEQ ID NO:1: -GGACTCACTGTATTCCACTGGCATCAA-3' SEQ ID NO:2: -TGAAGTGGAAAAAGGAGGAGGACAAGA -3'

Claims (19)

1. A method for the isolation of insulin-secreting cells from mammalian islet cells, said method comprising: culturing said islet cells under conditions which promote insulin secretion, and isolating those cells which produce one or more transcription factors associated with insulin production.

2. A method according to claim 1 wherein said transcription factor is selected from the group consisting of NKX 6.1, HNF3-beta, STF-1 and Beta-2.

3. A method according to claim 1 wherein said transcription factor is NKX 6.1.

4. A method according to claim 1 wherein said transcription factor is HNF3- beta. A method according to claim 1 wherein said transcription factor is STF-1.

6. A method according to claim 1 wherein said transcription factor is Beta-2.

7. A method for the isolation of insulin-secreting cells from mammalian islet 15 cells, substantially as hereinbefore described with reference to any one of the Examples.

8. Insulin-secreting cells produced by the method of any one of claims 1 to 7.

9. A method for proliferating insulin-secreting cells, said method comprising: .".culturing mammalian islet cells under conditions which promote insulin secretion, isolating those cells which produce one or more transcription factors associated with insulin production, and culturing the isolated cells under conditions which promote production of one or *o o: more of said transcription factors. A method according to claim 9 wherein said transcription factor is selected o. 25 from the group consisting of NKX 6.1, HNF3-beta, STF-1 and Beta-2.

11. A method according to claim 9 wherein said transcription factor is NKX 6.1.

12. A method according to claim 9 wherein said transcription factor is HNF3- beta.

13. A method according to claim 9 wherein said transcription factor is STF-1.

14. A method according to claim 9 wherein said transcription factor is Beta-2. A method for proliferating insulin-secreting cells, substantially as hereinbefore described with reference to any one of the Examples.

16. Insulin-secreting cells produced by the method of any one of claims 9 to

17. A method for optimizing the insulin-secreting ability of mammalian islet cells, said method comprising: culturing said islet cells under a variety of culturing conditions designed to promote insulin secretion, and selecting those conditions which stimulate production of elevated levels of one or _4o more transcription factors associated with insulin production. [N:/libc]00130:bmv W i 13

18. A method according to claim 17 wherein said transcription factor is selected from the group consisting of NKX 6.1, HNF3-beta, STF-1 and Beta-2.

19. A method according to claim 17 wherein said transcription factor is NKX 6.1. A method according to claim 17 wherein said transcription factor is HNF3- beta.

21. A method according to claim 17 wherein said transcription factor is STF-1.

22. A method according to claim 17 wherein said transcription factor is Beta-2.

23. A method for optimizing the insulin-secreting ability of mammalian islet cells, substantially as hereinbefore described with reference to any one of the Examples. Dated 20 January, 1999 VivoRx Pharmaceuticals, Inc. 000e Patent Attorneys for the Applicant/Nominated Person g"e SPRUSON FERGUSON *0 ooo*o 0 C O S C 00 [N:/libc]00130:bmv