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Dner-mediated cell purification of pancreatic endocrine pre-progenitor cells and their progeny

一种内分泌祖细胞、胰腺细胞的技术，应用在DNER介导的胰腺内分泌前祖细胞及其子代的细胞纯化领域，能够解决荧光增加等问题

Inactive Publication Date: 2011-03-02
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, addition of goat anti-Dner resulted in a significant increase in fluorescence, suggesting that Dner protein can be used as a tag in FACS

Method used

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  • Dner-mediated cell purification of pancreatic endocrine pre-progenitor cells and their progeny
  • Dner-mediated cell purification of pancreatic endocrine pre-progenitor cells and their progeny
  • Dner-mediated cell purification of pancreatic endocrine pre-progenitor cells and their progeny

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0227] Example 1: Bioinformatics Analysis of Mouse and Human Dner

[0228] To discover extracellular and transmembrane domains, mouse and human Dner were run through CBS's transmembrane predictor TMHMM server v.2.0 (http: / / www.cbs.dtu.dk / ). The results showed that both human and mouse Dner were predicted as single transmembrane proteins (type I), the main first 638 amino acids were on the extracellular side in both humans and mice, and the transmembrane domain was predicted to be amino acids 639-661. The transmembrane-like region predicted by TMHMM in the first part of the Dner protein is the signal peptide, which is also discussed below in the results related to SignalP.

[0229] Dner's mouse and human signal peptides were analyzed using CBS's SignalP 3.0 prediction server (http: / / www.cbs.dtu.dk / ). Mouse: signal peptide probability: 1.000; signal anchor probability: 0.000; maximum cleavage site probability: 0.494, between positions 39 and 40. Human: signal peptide probabili...

Embodiment 2

[0234] Example 2: Expression of Dner and Ddr1 in pancreatic beta cell lines

[0235]Total RNA was isolated from mouse endocrine-like cell lines: Ins1, βHC, βTCtet (condition: cultured with tetracycline (+tet)), βTCtet (condition: cultured without tetracycline (-tet)), Min6, αTC, and isolated from e15. 5 NMRI gastrointestinal tract (stomach, duodenum, spleen and pancreas) tissue. The cell line Ins1 is described in M ​​Asfari et al., Endocrinology, Vol. 130, 167-178. The cell line BetaHC-9 is described in Noda M et al., Diabetes, 1996 Dec;45(12):1766-73. The cell line βTC-tet is described in Fleischer N et al., Diabetes, 1998 Sep;47(9):1419-25. The cell line Min6 is described in J Miyazaki et al., Endocrinology, 1990 Jul;127(1):126-32. The cell line αTC is described in Powers AC et al., Diabetes, 1990 Apr;39(4):406-14. Prepare randomly primed cDNA using RNA as template. These cDNAs were subjected to 35 rounds of PCR (96°C, 180 seconds, then (96°C, 30 seconds, 55°C, 30 secon...

Embodiment 3

[0237] Example 3: Expression of Dner and Ddr1 in the endocrine-like cell line αTC, visualized by IHC in chamber slides

[0238] αTC was grown in DMEM containing 1000 mg / L D-glucose, sodium pyruvate (0.5 mM), P / S (penicillin (10 IU / ml), streptomycin (10 μg / ml)) and 10% FCS. The cell line αTC is described in Powers AC et al., Diabetes, 1990 Apr;39(4):406-14. Cells were fixed with Lilliys fixative for 45 minutes, then washed with PBS and stored in PBS at +5°C until use. Infiltrate by ethanol gradient (start at 70%, change to 96%, then change to 99%, then change to 99%, then change to 96%, finally change to 70%, use 5 min incubation for each concentration) Cells were then blocked with 0.5% TNB blocking reagent (from Perkin-Elmer) and stained. Goat anti-Dner (R&D Systems, AF2254, 1:150) or mouse anti-Ddr1 (R&D Systems, MAB2396, 1:50) was then added and incubated overnight (O / N) at room temperature (RT). The next day, cells were washed with PBS and biotinylated anti-donkey or ant...

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Abstract

The invention relates to a method of identifying and obtaining a culture cells comprising cells selected from the group consisting of endocrine pre-progenitor cells, endocrine progenitor cells, early endocrine cells, and / or fully differentiated endocrine cells. Also contemplated is a method of expanding the number of such cells as well as sorting such cells. In some aspects the invention further relates to a selective cell surface marker, DNER, that permits the selection of a unique subset of cells with endocrine pre-progenitor, endocrine progenitor, early endocrine, and / or fully differentiated endocrine phenotype. In some aspects the selective cell surface marker is selected from the group consisting of DNER, DISP2, SEZ6L2, LRP1 1 and SLC30A8. Furthermore, the invention relates to isolated cells selected from such cells and compositions thereof.

Description

field of invention [0001] In certain aspects, the invention relates to a selective cell surface marker, DNER, which enables the identification, selection and / or quantification of distinct cell subclasses and their progeny with a pancreatic endocrine pre-progenitor phenotype. In certain aspects, the selectable cell surface marker is selected from DNER, DISP2, SEZ6L2, LRP11 and SLC30A8. [0002] Compositions, cell cultures and cell populations comprising pancreatic cells selected from endocrine pre-progenitor cells, endocrine progenitor cells, early endocrine cells and / or fully differentiated endocrine cells, and the production of fully differentiated endocrine cells are also contemplated. Cells and methods for detecting endocrine pre-progenitor cells, endocrine progenitor cells, early endocrine cells and / or fully differentiated endocrine cells. Background of the invention [0003] Beta-cell transplantation has the potential to improve the treatment of type 1 diabetes, but ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N33/569
CPCG01N33/56966A61P1/18A61P3/10A61P5/48A61P5/50
Inventor J·哈尔德O·D·马德森G·格拉德沃尔G·梅利特泽
Owner NOVO NORDISK AS
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