title: Morphine sulfate concomitantly decreases neuronal differentiation and opioid receptor expression in mouse embryonic stem cells.
abstract: Opioids have been shown to affect prenatal and postnatal neural development in mammals. The present study investigates the impact of morphine sulfate (MS) treatment on neuronal differentiation as well as μ-opioid receptor (MOR) expression in mouse embryonic stem (mES) cells. Stem cells were manipulated in culture to differentiate in 3 sequential stages: Stage 1, cell transformation to embryoid bodies (EB); Stage 2, EB cell differentiation to neural progenitor (NP) cells; and, Stage 3, NP cell differentiation to neurons/astrocytes co-cultured cells. Using RT-PCR and flow cytometry analyses, cell types were confirmed by monitoring expression of Oct4, nestin, microtubule-associated protein 2 (mtap-2), and glial fibrillary acidic protein (GFAP) as cell-specific markers for stem cells, NP cells, neurons, and astrocytes, respectively. Similarly, gene expression for MOR, κ-opioid receptor (KOR), and δ-opioid receptor (DOR) was confirmed in each cell type. In order to investigate the effects of MS on differentiation, cells were treated with MS (1, 10, 100 μM) at either early (Stage 1) or late (Stage 3) stage of cellular differentiation. At Stage 1 exposure, MOR gene expression and neuroectoderm specific marker expression of nestin were down-regulated in both EB and NP cells. In addition, the opioid down-regulated GFAP in differentiated neurons/astrocytes co-cultured cells. Late stage treatment with MS resulted in a down-regulation of mtap-2 and GFAP in differentiated neurons/astrocytes co-cultured cells. Moreover, late stage treatment with MS and naltrexone inhibited the effect of MS on neuronal differentiation, suggesting that MS treatment interferes with differentiation via MOR activation. Together, the results show that MS exposure at early and late stage of cellular differentiation significantly decreases genotype and phenotype in differentiated neuronal cells. The results of this study have implications regarding the potential effect of opiates on fetal brain development.
authors.affiliation: St. John's University, College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences, Queens, NY 11439, United States.
authors.forename: Sanjay L
authors.initials: SL
authors.lastname: Dholakiya
authors.affiliation: St. John's University, College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences, Queens, NY 11439, United States.
authors.forename: Angela
authors.initials: A
authors.lastname: Aliberti
authors.affiliation: St. John's University, College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences, Queens, NY 11439, United States. Electronic address: barilef@stjohns.edu.
authors.forename: Frank A
authors.initials: FA
authors.lastname: Barile
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D018920:Coculture Techniques; D015536:Down-Regulation; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009020:Morphine / Q000494:pharmacology*; D000066450:Mouse Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D064231:Nestin / Q000235:genetics / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology / Q000187:drug effects; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*
chemical_list: D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; C577485:Nes protein, mouse; D064231:Nestin; D050814:Octamer Transcription Factor-3; C404943:Oprm protein, mouse; C494174:Pou5f1 protein, mouse; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; C528900:glial fibrillary astrocytic protein, mouse; D009020:Morphine
keywords: Morphine; Mouse embryonic stem cells; Neuronal differentiation; Opioid receptors