Nicotine exposure during differentiation causes inhibition of N-myc expression.
BACKGROUND
The ability of chemicals to disrupt neonatal development can be studied using embryonic stem cells (ESC). One such chemical is nicotine. Prenatal nicotine exposure is known to affect postnatal lung function, although the mechanisms by which it has this effect are not clear. Since fibroblasts are a critical component of the developing lung, providing structure and secreting paracrine factors that are essential to epithelialization, this study focuses on the differentiation of ESC into fibroblasts using a directed differentiation protocol.


METHODS
Fibroblasts obtained from non-human primate ESC (nhpESC) differentiation were analyzed by immunohistochemistry, immunostaining, Affymetrix gene expression array, qPCR, and immunoblotting.


RESULTS
Results of these analyses demonstrated that although nhpESCs differentiate into fibroblasts in the presence of nicotine and appear normal by some measures, including H&E and SMA staining, they have an altered gene expression profile. Network analysis of expression changes demonstrated an over-representation of cell-cycle related genes with downregulation of N-myc as a central regulator in the pathway. Further investigation demonstrated that cells differentiated in the presence of nicotine had decreased N-myc mRNA and protein expression and longer doubling times, a biological effect consistent with downregulation of N-myc.


CONCLUSIONS
This study is the first to use primate ESC to demonstrate that nicotine can affect cellular differentiation from pluripotency into fibroblasts, and in particular, mediate N-myc expression in differentiating ESCs. Given the crucial role of fibroblasts throughout the body, this has important implications for the effect of cigarette smoke exposure on human development not only in the lung, but in organogenesis in general.
Ahmi
A
Ben-Yehudah
Becki M
BM
Campanaro
Laura M
LM
Wakefield
Tia N
TN
Kinney
Jill
J
Brekosky
Vonya M
VM
Eisinger
Carlos A
CA
Castro
Division of Developmental and Regenerative Medicine, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA 15213, USA. Dlc4@pitt.edu.
Diane L
DL
Carlisle
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D005347:Fibroblasts / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D066298:In Vitro Techniques; D008168:Lung / Q000196:embryology / Q000378:metabolism / Q000473:pathology; D023421:Models, Animal; D009538:Nicotine / Q000494:pharmacology*; D011323:Primates; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism
D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D009538:Nicotine