Transcript 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.

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
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)
Gastrointestinal Neuroendocrine Cells
 1-2% by volume of mucosa
 Sequestrated in crypts within the mucosa
Difficult cells to isolate and examine
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
Characteristics potentially useful for FACS
 Size
 Density
 Acidic vesicles
 Vesicular monoamine transporters (VMAT)
 Acid gradient
 Accumulates weak bases
[H+]
V-type
ATPase
[Amine]
[H+]
pH
VMAT
1/2
[Amine]
Vesicles accumulate weak bases
Acridine Orange
→
Cytoplasmic RNA →
Nuclei
fluoresce green
fluoresce orange
Absorption
Emission
FITC/Cy7 channel
AO widely used as a pH-sensitive dye in studies of acid secretion
Acridine Orange
Parietal cells
Neuroendocrine cells
pH 1-2
pH 3-5
AO Accumulation
Stacking
AO Accumulation
No stacking
pH determines emission
Orange/Red
Acridine Orange – Gastric mucosa FACS
Parietal cells*
Neuroendocrine
ECL cells*
*95-99%
Green
pure
Lambrecht N et al.
Physiol Genomics 2006; 25:153-65
Rodent gastric cell populations separated by AO fluorescence
Results – Human Gastric ECL cells
97.3-99.1% pure
(HDC-positive)
ECL
• 92.9-95.6% viable
• Proliferate in short-term culture
Human gastric neuroendocrine ECL cells separated by AO fluorescence
Protocol developed for Small Intestinal EC cells
FACS approach
Mixed cell
population
used for control
studies
Terminal ileum
Mixed cell population
F0 (~4%EC cells)
Collagenase/pronase digestion
of tissue at 37OC for 1 hour
Nycodenz gradient centrifugation
FN (~75% pure EC cells)
1.07 g/l
Confirm by EM/confocal microscopy,
immunostaining and PCR of
neuroendocrine markers, measure
serotonin content
Immunostaining of FN
with acridine orange
99% Pure live EC cell preparation
~ 1 million cells
FACS of live EC cells
Kidd M. et al.
Am J Physiol Gastrointest Liver Physiol. 2006 Feb 2; [Epub ahead of print]
FACS sorting – Human Small Intestinal Mucosa
[AO] =50-200nM
1% of nuclear stain
Results – Human Small Intestinal EC cells
A
B
Human EC Cell preps (n = 4)
Ileal Mucosa (F0)
Nycodenz (FN)
FACS-AO
2-fold
28-fold
67-fold
TPH +ve cells (%)
4.2±0.6
75±8.2
990.9
CgA +ve cells (%)
6.3±1.1
84±3
1001.3
2.8 X 107
2.7 X 106
7.2 X 105
99.6
97.9
99.3
5-HT (compared to mucosa)
Cell Number*
Viability (%) (Trypan Blue)
99% preparations of naïve human EC cells
Modlin I.M. et al.
J Clin Endocrinol Metab. 2006 Mar 14; [Epub ahead of print]
Secretion – Human Small Intestinal EC cells
Forskolin
EC50 = 2.1x10-7M
Isoproterenol
EC50 = 8.1x10-8M
Short-term culture
Serotonin secretion
cAMP/adrenergic control
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
Future Directions
•
Define cellular regulators = Understand physiology
= Unravel pathobiology
= Identify new therapeutic targets
Acknowledgements
Irvin Modlin
GI Pathobiology Research Group
Manish Champaneria
Geeta Eick
Dept. Surgery, Yale
Robert Camp
Pathology, Yale
Shrikant Mane
Keck, Yale
Geoff Lyon
Mark Shlomchik
FACS, Yale
George Sachs
Nils Lambrecht
Physiology, UCLA