1. Use of FACS in the Isolation and Characterization of Gastrointestinal Neuroendocrine Cells Mark Kidd, Ph.D. GI Surgical Pathobiology Research Group (Irvin Modlin) Department of Surgery

2. Gastrointestinal Neuroendocrine cell Neoplasia “Carcinoid”  Tumor incidence has increased 700-2700% since 1983 *  Little is known about the physiology or pathobiology  No significant advances in therapeutic modalities  Neuroendocrine cells = Progenitor cells of neoplasia  No pure naïve neuroendocrine cell preparation *NCI (1973-2002)

3.  1-2% by volume of mucosa  Sequestrated in crypts within the mucosa Gastrointestinal Neuroendocrine Cells Difficult cells to isolate and examine

4. Previous protocols for neuroendocrine cell isolation  Mucosal scrapping or inverted mucosal sacs  Digestion with pronase/collagenase  Respiration/calcium-free media = Cell slurry ~1-2% pure neuroendocrine cells  Nycodenz gradient centrifugation  Elutriation  Short-term culture 50-70% 72-84% 80-90% Enrichment Significant enrichment but not homogeneous

5. Characteristics potentially useful for FACS  Size  Density  Acidic vesicles  Vesicular monoamine transporters (VMAT)  Acid gradient  Accumulates weak bases V-type ATPase [H + ] [Amine] VMAT 1/2 pH Vesicles accumulate weak bases

6. Acridine Orange AbsorptionEmission FITC/Cy7 channel Nuclei → fluoresce green Cytoplasmic RNA → fluoresce orange AO widely used as a pH-sensitive dye in studies of acid secretion

7. Acridine Orange Parietal cells Neuroendocrine cells pH 1-2pH 3-5 AO Accumulation Stacking AO Accumulation No stacking pH determines emission

8. Acridine Orange – Gastric mucosa FACS Orange/Red Green Parietal cells * Neuroendocrine ECL cells * Rodent gastric cell populations separated by AO fluorescence * 95-99% pure Lambrecht N et al. Physiol Genomics 2006; 25:153-65

9. Results – Human Gastric ECL cells ECL Human gastric neuroendocrine ECL cells separated by AO fluorescence 97.3-99.1% pure (HDC-positive) 92.9-95.6% viable Proliferate in short-term culture

10. Protocol developed for Small Intestinal EC cells FACS approach Terminal ileum Collagenase/pronase digestion of tissue at 37 O C for 1 hour Mixed cell population F 0 (~4%EC cells) Nycodenz gradient centrifugation F N (~75% pure EC cells) Immunostaining of F N with acridine orange FACS of live EC cells 99% Pure live EC cell preparation ~ 1 million cells 1.07 g/l Mixed cell population used for control studies Confirm by EM/confocal microscopy, immunostaining and PCR of neuroendocrine markers, measure serotonin content Kidd M. et al. Am J Physiol Gastrointest Liver Physiol. 2006 Feb 2; [Epub ahead of print]

11. FACS sorting – Human Small Intestinal Mucosa [AO] =50-200nM 1% of nuclear stain

12. Results – Human Small Intestinal EC cells Human EC Cell preps (n = 4) Ileal Mucosa (F 0 )Nycodenz (F N )FACS-AO 5-HT (compared to mucosa)2-fold28-fold67-fold TPH +ve cells (%)4.2±0.675±8.2 99  0.9 CgA +ve cells (%)6.3±1.184±3 100  1.3 Cell Number*2.8 X 10 7 2.7 X 10 6 7.2 X 10 5 Viability (%) (Trypan Blue)99.697.999.3 AB 99% preparations of naïve human EC cells Modlin I.M. et al. J Clin Endocrinol Metab. 2006 Mar 14; [Epub ahead of print]

13. Secretion – Human Small Intestinal EC cells EC 50 = 2.1x10 -7 M EC 50 = 8.1x10 -8 M Short-term culture Serotonin secretion cAMP/adrenergic control Forskolin Isoproterenol

14. Summary Method established for gastric ECL cells Small intestinal EC cells can be isolated by similar approach Viable, highly purified preparations Short-term culture Proliferation/secretory studies Transcriptome analysis Define cellular regulators = Understand physiology = Unravel pathobiology = Identify new therapeutic targets Future Directions

15. Acknowledgements Manish Champaneria Geeta Eick Robert Camp Shrikant Mane Geoff Lyon Mark Shlomchik George Sachs Nils Lambrecht Dept. Surgery, Yale Pathology, Yale Keck, Yale FACS, Yale Physiology, UCLA GI Pathobiology Research Group Irvin Modlin