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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0676—Pancreatic cells

Definitions

• 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 .
• invention methods result in the expression of certain characteristic intracellular proteins (e.g., HNF3 beta, NKX 6.1, STF-1, or beta2) whose activity regulates the transcription of the insulin gene.
• 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-and- pump. This treatment, however, can only temporarily delay the pathological complications of the disease.
• Fetal pancreatic islets contain many beta stem cells which can mature after transplantation and which are 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) .
• these pancreatic islet cells may retain regulatory pathways which originated in the gut to direct expression of the same peptide hormone genes.
• 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.
• tissue-specific promoter element TSE
• 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)] .
• 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) ] .
• 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 (e.g., HNF3- beta, STF-l, NKX 6.1 or Beta2) which exert direct effects upon the insulin promoter and thereby alter its tran ⁇ criptional activity.
• HNF3- beta a transcription factor that exert direct effects upon the insulin promoter and thereby alter its tran ⁇ criptional 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.
• methods for the isolation of insulin-secreting cells from mammalian islet cells 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.
• Ham's F12 medium GibcoBRL #317865-035
• fetal bovine serum in the range of about 2-4% fetal bovine serum and in the range of about 10-200 ⁇ g/ml of bovine pituitary extract
• 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 ⁇ g/ml of apotransferrin, 20-500 ⁇ g/ml of hypothalamic extract, 0-2% bovine serum albumin, and the like.
• 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.
• 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 "t.issue-s_pecific promoter element (s) " (TSEs) promixal to the insulin promoter.
• TSEs tissue-s_pecific promoter element promixal to the insulin promoter.
• the transcription factors have the ability to transactivate the gene expression of pancreatic hormones.
• the transcription factor STF-l (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, e.g., NKX 6.1, HNF3-beta, STF-l, Beta-2, and the like.
• 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.
• STF-l 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-l 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-l activity, but also provides a probe for identifying STF-l cells, and thus insulin-producing cells.
• STF-l is a member of the homeobox class of transcription factors and is required for pancreatic organogenesis [Johnson, et al . , NATURE 371: 606-609
• STF-l In adult beta and delta cells, STF-l is required for the hallmark phenotype of these cells, the expression of insulin in beta cells [Peers et al . , MOLECULAR ENDOCRINOLOGY 7:1798-1806 (1994)] and somatostatin in delta cells [Leonard et al . , MOLECULAR ENDOCRINOLOGY 7:1275-1283 (1993)] .
• STF-l binds to the CT2 box in the human insulin promoter [Petersen et al. , PROC. NAT. ACAD. SCI. USA 91:10465-10469 (1994)] resulting in increased transcription of the insulin gene.
• transcription factors e.g., STF-l, HNF3 beta, NKX 6.1, Beta2, and the like
• STF-l can be utilized as both markers for mature islet cells and as a requirement for insulin expression.
• HNF3 beta can be utilized as both markers for mature islet cells and as a requirement for insulin expression.
• culture conditions can be optimized based upon the expression levels and activities of such transcription factors in conjunction with measurements of glucose-responsive insulin release.
• methods for proliferating insulin-secreting cells 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.
• methods for optimizing the insulin-secreting ability of mammalian islet cells 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.
• 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 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-l, HNF3 beta, NKX 6.1, and Beta2 protein levels as determined by Western blot analysis.
• the presence of STF-l 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°C with 0.5 ml each Cell Lysis Solution (1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM 2- mercaptoethanol, 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-l specific antibody [Peers et al.
• This example describes the quantitation of STF-l 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-l mRNA are determined by quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) utilizing primers that distinguish the correctly spliced message, e.g.,
• This example demonstrates the use of the level and activity of the STF-l protein to optimize beta cell culture medium.
• the interaction of STF-l with the CT2 element in the insulin promoter is augmented in insulinoma cells following glucose stimulation implying that both the level and the state (phosphorylation, glycosylation etc.) of the STF-l 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-l protein.
• the levels of STF-l 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.
• STF-l protein levels normalized for cell number remain constant.
• the glucose-dependent modification of STF-l 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 . This is the first report of the use of the state and level of STF-l protein to optimize beta cell culture conditions.
• islet cells have been identified in representative cultures by in situ hybridization with a STF-l probe.
• Co-localization of STF-l 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.
• This example describes means to regulate the STF-l promoter.
• High level expression of STF-l is believed to be required for the beta cell phenotype.
• 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-l promoter. Reporter gene expression data can be correlated with other data and conditions optimized for glucose-dependent insulin release.

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• 1997
  • 1997-06-24 JP JP10503496A patent/JP2000513227A/ja active Pending
  • 1997-06-24 AU AU34104/97A patent/AU724745B2/en not_active Ceased
  • 1997-06-24 EP EP97930221A patent/EP0929573A1/en not_active Withdrawn
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