Ethanol increases fetal human neurosphere size and alters adhesion molecule gene expression.
BACKGROUND
Ethanol (ETOH) consumption by pregnant women can result in Fetal Alcohol Spectrum Disorder (FASD). To date, the cellular targets and mechanisms responsible for FASD are not fully characterized. Our aim was to determine if ETOH can affect fetal human brain-derived neural progenitor cells (NPC).


METHODS
Neural progenitor cells were isolated by positive selection from normal second trimester fetal human brains (n = 4) and cultured, for up to 72 hours, in mitogenic media containing 0, 1, 10, or 100 mM ETOH. From 48 to 72 hours in culture, neurospheres generated in these conditions were filmed using time-lapse video microscopy. At the end of 72 hours, neurosphere diameter and roundness were measured using videographic software. Mitotic phase analysis of cell-cycle activity and apoptotic cell count were also performed at this time, by flow cytometry using propidium iodide (PI) staining. Real-time PCR was used to estimate expression of genes associated with cell adhesion pathways.


RESULTS
Neurosphere diameter correlated positively (r = 0.87) with increasing ETOH concentrations. There was no significant difference in cell-cycle activity and no significant increase in apoptosis with increasing ETOH concentrations. Time-lapse video microscopy showed that ETOH (100 mM) reduced the time for neurosphere coalescence. Real-time PCR analysis showed that ETOH significantly altered the expression of genes involved in cell adhesion. There was an increase in the expression of alpha and beta Laminins 1, beta Integrins 3 and 5, Secreted phosphoprotein1 and Sarcoglycan epsilon. No change in the expression of beta Actin was observed while the expression of beta Integrin 2 was significantly suppressed.


CONCLUSIONS
ETOH had no effect on NPC apoptosis but, resulted in more rapid coalescence and increased volume of neurospheres. Additionally, the expression of genes associated with cell adhesion was significantly altered. ETOH induced changes in NPC surface adhesion interactions may underlie aspects of neurodevelopmental abnormalities in FASD.
Vangipuram
Sharada D
SD
Children's Research Center of Michigan, The Carman and Ann Adams Department of Pediatrics, Wayne State University School of Medicine and Children's Hospital of Michigan, Detroit, MI 48201, USA. svangipuram@med.wayne.edu
Grever
William E
WE
Parker
Graham C
GC
Lyman
William D
WD
D017209:Apoptosis / Q000235:genetics; D001921:Brain / Q000187:drug effects / Q000473:pathology*; D002453:Cell Cycle / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000633:toxicity; D005260:Female; D063647:Fetal Alcohol Spectrum Disorders / Q000235:genetics* / Q000473:pathology; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005865:Gestational Age; D006801:Humans; D007231:Infant, Newborn; D039641:Integrin beta Chains / Q000235:genetics*; D039661:Integrin beta3 / Q000235:genetics*; D007797:Laminin / Q000235:genetics*; D018715:Microscopy, Video; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D009924:Organ Culture Techniques; D053495:Osteopontin / Q000235:genetics*; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction; D049031:Sarcoglycans / Q000235:genetics*; D013234:Stem Cells / Q000187:drug effects / Q000473:pathology*
D039641:Integrin beta Chains; D039661:Integrin beta3; C478221:LAMB1 protein, human; D007797:Laminin; C526572:SGCE protein, human; D049031:Sarcoglycans; C084344:integrin beta5; D053495:Osteopontin; C084098:laminin A; D000431:Ethanol