title: Effect of nicotine on the proliferation and chondrogenic differentiation of the human Wharton's jelly mesenchymal stem cells.
abstract: BACKGROUND
  Osteoarthritis (OA) is a chronic joint disease characterized by a progressive and irreversible degeneration of articular cartilage. Among the environmental risk factors of OA, tobacco consumption features prominently, although, there is a great controversy regarding the role of tobacco smoking in OA development. Among the numerous chemicals present in cigarette smoke, nicotine is one of the most physiologically active molecules.
  
  
  OBJECTIVE
  The aim of the study was (i) to measure the impact of nicotine on the proliferation and chondrogenic differentiation of mesenchymal stem cells from the human Wharton's jelly (hWJ-MSCs) into chondrocytes, (ii) to investigate whether the α7 nicotinic acetylcholine receptors (nAChRs) was expressed in hWJ-MSCs and could play a role in the process. The project benefits from the availability of an umbilical cord bank from which hWJ-MSCs were originated.
  
  
  METHODS
  The hWJ-MSCs were cultured and used up to passage 5. The proliferation of hWJ-MSCs with 5 μM nicotine was measured by the MTT assay on the 1st, 2nd, 3rd, and 6th day. Flow cytometry analysis was used to detect cell apoptosis/necrosis by Annexin V/PI double-staining. The chondrogenic differentiation grade of hWJ-MSCs induced by TGFβ3 was assessed by the Sirius red and Alcian blue staining. The expression of markers genes was followed by quantitative real-time PCR. The expression of nAChRs was followed by RT-PCR. The functional activity of α7 nAChR was evaluated by calcium (Ca2+) influx mediated by nicotine using the Fluo-4 NW Calcium assay.
  
  
  RESULTS
  The proliferation of hWJ-MSCs was significantly impaired by nicotine (5 μM) from the 3rd day of treatment, but nicotine did not significantly induce modifications on the viability of hWJ-MSCs. Alcian blue staining indicated that the amount of proteoglycan was more abundant in control group than in the nicotine group, but no difference was observed on the total collagen amount using Sirius red staining. The mRNA expression of Sox9, type II collagen (Col2a1), aggrecan in control group was higher than in the nicotine group. We found that hWJ-MSCs expressed α7 nAChR. The receptor agonist nicotine caused calcium (Ca2+) influx into hWJ-MSCs suggesting that the calcium ion channel α7 homopolymer could mediate this response.
  
  
  CONCLUSIONS
  At the concentration used, nicotine had an adverse effect on the proliferation and chondrogenic differentiation of hWJ-MSCs which was probably impaired through a α7 nAChR mediation.
authors.affiliation: UMR 7365 CNRS-UL, Faculté de Médecine, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), 54505 Vandœuvre-lès-Nancy, France.|Department of Orthopaedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
authors.forename: Yongjian
authors.initials: Y
authors.lastname: Qi
authors.affiliation: UMR 7365 CNRS-UL, Faculté de Médecine, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Léonore
authors.initials: L
authors.lastname: Avercenc-Leger
authors.affiliation: UMR 7365 CNRS-UL, Faculté de Médecine, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Vincourt
authors.affiliation: Faculté de Médecine, Bio-ingénierie Moléculaire Cellulaire et Thérapeutique FR3209 CNRS, Campus Biologie Santé, 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Hupont
authors.affiliation: UMR 7365 CNRS-UL, Faculté de Médecine, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Huselstein
authors.affiliation: Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071, China.|Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Orthopaedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
authors.forename: Liaobin
authors.initials: L
authors.lastname: Chen
authors.affiliation: UMR 7365 CNRS-UL, Faculté de Médecine, Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), 54505 Vandœuvre-lès-Nancy, France.
authors.forename: Jacques
authors.initials: J
authors.lastname: Magdalou
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects*; D009538:Nicotine / Q000009:adverse effects*; D018722:Nicotinic Agonists / Q000009:adverse effects*; D010003:Osteoarthritis / Q000209:etiology / Q000378:metabolism; D012907:Smoking / Q000009:adverse effects; D059631:Wharton Jelly / Q000166:cytology*; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000032:analysis / Q000378:metabolism
chemical_list: D018722:Nicotinic Agonists; D064569:alpha7 Nicotinic Acetylcholine Receptor; D009538:Nicotine
keywords: Nicotine; chondrogenic differentiation; human Wharton’s jelly mesenchymal stem cells; nicotinic acetylcholine receptor; osteoarthritis