In vitro characteristics of pulmonary neuroendocrine cells isolated from rabbit fetal lung. I. Effects of culture media and nerve growth factor.
Pulmonary neuroendocrine (NE) cells, dispersed throughout the airway mucosa as single cells and as innervated clusters (neuroepithelial bodies), were isolated from rabbit fetal lung and studied in short-term culture. The effects of culture media and nerve growth factor (NGF) on in vitro maintenance, differentation, and cell kinetics of isolated NE cells were examined. For demonstration of NE cells in intact lung, during cell separation and after culture, immunostaining for serotonin, formaldehyde-induced fluorescence method, histochemical reaction for acetylcholinesterase, and electron microscopy were used. The isolation procedure consisted of mechanical and enzymatic dissociation of lung tissue followed by separation of isolated cells on a discontinuous gradient of Percoll, resulting in 5- to 10-fold enrichment in NE cells. Cell fractions enriched in NE cells were cultured up to 7 days either in supplemented alpha-minimal essential medium with fetal bovine serum or in defined, hormone-supplemented, serum-free medium. NGF (2.5 S 5 to 50 ng/ml) was added to both serum-supplemented and serum-free media; cultures without NGF served as control. The number of serotonin-immunoreactive NE cells maintained in serum-supplemented medium (0.5% fetal bovine serum) increased significantly (p less than 0.05) on days 4 and 7 compared with cultures grown in serum-free medium. NE cells maintained in serum-supplemented medium incorporated [3H]thymidine and their labeling index was significantly increased (p less than 0.01) on day 7, whereas few or no NE cells were labeled in cultures grown in serum-free medium. NGF had no effect on the maintenance or kinetics of NE cells. Cultured NE cells formed elongated (unipolar or bipolar) neurite-like cytoplasmic processes with a button-like ending, regardless of the presence of NGF. Amine accumulated in perinuclear cytoplasm and in button-like endings. Staining for acetylcholinesterase (strongly positive in intact neuroepithelial bodies) was not detectable after separation into single cells, culture, or exposure to NGF. This study demonstrates that NE cells can be isolated from rabbit fetal lung and maintained in short-term culture. Low concentrations of fetal bovine serum enhanced the in vitro maintenance of NE cells, whereas NGF had no such effect. A feeder layer may be also important, since NE cells were closely associated with lung epithelial or fibroblast-like cells. The formation of neurite-like processes appears to be an expression of paracrine/paraneuron-type cell differentiation not mediated by NGF.(ABSTRACT TRUNCATED AT 400 WORDS)
Cutz
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E
Yeger
H
H
Wong
V
V
Bienkowski
E
E
Chan
W
W
D001078:APUD Cells / Q000166:cytology / Q000648:ultrastructure; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D007124:Immunoenzyme Techniques; D008168:Lung / Q000166:cytology* / Q000196:embryology / Q000648:ultrastructure; D008854:Microscopy, Electron; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009490:Neurosecretory Systems / Q000166:cytology* / Q000196:embryology / Q000648:ultrastructure; D011817:Rabbits; D012701:Serotonin / Q000378:metabolism
D003470:Culture Media; D009414:Nerve Growth Factors; D012701:Serotonin