Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose...
-
Upload
teresa-garrison -
Category
Documents
-
view
216 -
download
0
Transcript of Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose...
![Page 1: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/1.jpg)
Small molecule-induced pancreatic exocrine transdifferentiation:
Assay development
Kristin RoseBridget Wagner, Ph.D.
Broad Chemical Biology, Broad Institute of MIT & HarvardSummer 2008
![Page 2: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/2.jpg)
Pancreas
• Endocrine
– Islets of Langerhans – large spherical cellular clusters
– Secrete hormones – glucagon – insulin– somatostatinPP – pancreatic polypeptide
• Exocrine
– Acini – small berry-like clusters
– Secrete digestive enzymes to intestine via ducts (trypsin, chymotrypsin…)
![Page 3: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/3.jpg)
Type 1 Diabetes
• Autoimmune – permanent destruction of
insulin-producing β cells
• No cure, lethal without insulin injections– Islet transplantation = last resort,
but risk of rejection, requires lifelong
immunosuppresant use
• So what can we do?
![Page 4: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/4.jpg)
Transdifferentiation
• Process of switching between
differentiated states
• Goal: Increase β cell mass
• Exocrine insulin-secreting?
![Page 5: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/5.jpg)
AR42J & ARIP
Commercially available rat pancreatic exocrine tumor cell lines
AR42J = acinar; ARIP = ductal
Project Cell Lines
![Page 6: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/6.jpg)
Project Cell Lines
• Why?
– Research literature shows these cell lines being induced to express insulin by various treatments, including conophylline
• Natural product-derived small molecule!• Encouragement to develop an assay
![Page 7: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/7.jpg)
Assay Development 1: qPCR
![Page 8: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/8.jpg)
Preliminary Results
• Establish basal levels of expression
• Quantitative comparison of untreated AR42J cells to INS-1 cells – INS-1 is a β cell-derived rat
tumor cell line (insulinoma)
Gene Fold difference of INS-1 to AR42J
Insulin 7786
Gcg --
PP 0.38
Pdx1 2771.91
Glut2 250.73
MafA 68.12
Nkx2.2 47.95
Pax4 15.69
Beta2 11.52
![Page 9: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/9.jpg)
Testing to identify a positive control
• Hepatocyte Growth Factor (HGF)– Literature-based positive control
• Activin A– Peptide (TGF- family) with role in endocrine function
• Glucagon-Like Peptide-1 (GLP-1)– Promotes cell function
• Exendin-4– Peptide analog of GLP-1, isolated from saliva of the Gila
monster, a poisonous lizard
• Trichostatin A (TSA)– Small-molecule histone deacetylase (HDAC) inhibitor
![Page 10: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/10.jpg)
Treatment effects on insulin gene expression in AR42J cells
0
1
2
3
4
5
NT HGF+Act GLP-1 exen-4
Treatment
Fo
ld C
han
ge
![Page 11: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/11.jpg)
Assay Development 2: Insulin protein expression
MIN6 cells (mouse beta)
(1°)
(2°)
nuclei
insulin
![Page 12: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/12.jpg)
Untreated AR42J Untreated ARIP
Assay Development 2: Insulin protein expression
nuclei
insulin
![Page 13: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/13.jpg)
Summary and future directions
• Plan: Screen for compounds to induce insulin expression in exocrine cells• First step: Assay development and identification of positive controls
• Approach 1: gene expression• HGF+activin and exendin-4 increase ins mRNA in AR42J• Need to optimize TSA concentration• Need to optimize GLP-1 treatment
• Approach 2: protein expression• Immunofluorescence-based approach• Ideal for high-throughput screening• But more stringent screening condition (all the way to protein)• Working on positive control conditions
![Page 14: Small molecule-induced pancreatic exocrine transdifferentiation: Assay development Kristin Rose Bridget Wagner, Ph.D. Broad Chemical Biology, Broad Institute.](https://reader035.fdocuments.us/reader035/viewer/2022062518/56649e905503460f94b95313/html5/thumbnails/14.jpg)
Acknowledgements
Broad Chemical BiologyDeepika Walpita
Stefan Kubicek
Tammy Gilbert
Dina Fomina
Yuan Yuan
Danny Chou
Alex Gitlin
Yiying Xu
Bridget Wagner
Stuart Schreiber
Broad/Summer Research Program in GenomicsMaura Silverstein
Lucia Vielma
Shawna Young
Bruce Birren
Eric Lander