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)

1-2% by volume of mucosa

Sequestrated in crypts within the mucosa

Gastrointestinal Neuroendocrine Cells

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

V-type ATPase[H+] [H+]

[Amine][Amine]VMAT

1/2

pH

Vesicles accumulate weak bases

Acridine Orange

Absorption Emission

FITC/Cy7 channel

Nuclei → fluoresce green

Cytoplasmic RNA → fluoresce orange

AO widely used as a pH-sensitive dye in studies of acid secretion

Acridine Orange

Parietal cells Neuroendocrine cells

pH 1-2 pH 3-5AO Accumulation

StackingAO Accumulation

No stacking

pH determines emission

Acridine Orange – Gastric mucosa FACS

Ora

nge/

Red

Green

Parietal cells*

NeuroendocrineECL cells*

Rodent gastric cell populations separated by AO fluorescence

*95-99% pureLambrecht N et al.

Physiol Genomics 2006; 25:153-65

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

Protocol developed for Small Intestinal EC cells

FACS approach

Terminal ileum

Collagenase/pronase digestion of tissue at 37OC for 1 hour

Mixed cell populationF0 (~4%EC cells)

Nycodenz gradient centrifugationFN (~75% pure EC cells)

Immunostaining of FN

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]

FACS sorting – Human Small Intestinal Mucosa

[AO] =50-200nM1% of nuclear stain

Results – Human Small Intestinal EC cells

Human EC Cell preps (n = 4)

Ileal Mucosa (F0) Nycodenz (FN) FACS-AO

5-HT (compared to mucosa) 2-fold 28-fold 67-fold

TPH +ve cells (%) 4.2±0.6 75±8.2 990.9

CgA +ve cells (%) 6.3±1.1 84±3 1001.3

Cell Number* 2.8 X 107 2.7 X 106 7.2 X 105

Viability (%) (Trypan Blue) 99.6 97.9 99.3

A B

99% preparations of naïve human EC cellsModlin I.M. et al.

J Clin Endocrinol Metab. 2006 Mar 14; [Epub ahead of print]

Secretion – Human Small Intestinal EC cells

EC50 = 2.1x10-7M EC50 = 8.1x10-8M

Short-term cultureSerotonin secretion

cAMP/adrenergic control

Forskolin Isoproterenol

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

Acknowledgements

Manish ChampaneriaGeeta Eick

Robert Camp

Shrikant Mane

Geoff LyonMark Shlomchik

George SachsNils Lambrecht

Dept. Surgery, Yale

Pathology, Yale

Keck, Yale

FACS, Yale

Physiology, UCLA

GI Pathobiology Research GroupIrvin Modlin