Use of FACS

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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
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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)
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Gastrointestinal Neuroendocrine Cells

• 1-2 by volume of mucosa
• Sequestrated in crypts within the mucosa

Difficult cells to isolate and examine
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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
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Characteristics potentially useful for FACS

• Size
• Density
• Acidic vesicles

• Vesicular monoamine transporters (VMAT)
• Acid gradient
• Accumulates weak bases

Amine
Amine
VMAT 1/2
H
V-type ATPase
H
pH
Vesicles accumulate weak bases
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Acridine Orange
Nuclei ? fluoresce green Cytoplasmic RNA ?
fluoresce orange
Absorption
Emission
FITC/Cy7 channel
AO widely used as a pH-sensitive dye in studies
of acid secretion
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Acridine Orange
Parietal cells
Neuroendocrine cells
pH 1-2
pH 3-5
AO Accumulation Stacking
AO Accumulation No stacking
pH determines emission
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Acridine Orange Gastric mucosa FACS
Parietal cells
Orange/Red
Neuroendocrine ECL cells
95-99 pure Lambrecht N et al. Physiol Genomics
2006 25153-65
Green
Rodent gastric cell populations separated by AO
fluorescence
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Results Human Gastric ECL cells
97.3-99.1 pure (HDC-positive)

• 92.9-95.6 viable
• Proliferate in short-term culture

Human gastric neuroendocrine ECL cells separated
by AO fluorescence
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Protocol developed for Small Intestinal EC
cells FACS approach
Mixed cell population used for control studies
Terminal ileum
Mixed cell population F0 (4EC cells)
Collagenase/pronase digestion of tissue at 37OC
for 1 hour
Nycodenz gradient centrifugation FN (75 pure EC
cells)
1.07 g/l
Immunostaining of FN with acridine orange
99 Pure live EC cell preparation 1 million
cells
Confirm by EM/confocal microscopy, immunostaining
and PCR of neuroendocrine markers, measure
serotonin content
FACS of live EC cells
Kidd M. et al. Am J Physiol Gastrointest Liver
Physiol. 2006 Feb 2 Epub ahead of print
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FACS sorting Human Small Intestinal Mucosa
AO 50-200nM 1 of nuclear stain
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Results Human Small Intestinal EC cells
A
B
99 preparations of naïve human EC cells
Modlin I.M. et al. J Clin Endocrinol Metab. 2006
Mar 14 Epub ahead of print
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Secretion Human Small Intestinal EC cells
Isoproterenol
Forskolin
EC50 8.1x10-8M
EC50 2.1x10-7M
Short-term culture Serotonin secretion cAMP/adrene
rgic control
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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
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Acknowledgements
Irvin Modlin
GI Pathobiology Research Group
Dept. Surgery, Yale Pathology, Yale Keck,
Yale FACS, Yale Physiology, UCLA
Manish Champaneria Geeta Eick Robert
Camp Shrikant Mane Geoff Lyon Mark
Shlomchik George Sachs Nils Lambrecht
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