title: Muscle-derived stem cells seeded into acellular scaffolds develop calcium-dependent contractile activity that is modulated by nicotinic receptors.
abstract: OBJECTIVE
  To explore the contractile activity and physiologic properties of muscle-derived stem cells (MDSCs) incorporated into small intestinal submucosa (SIS) scaffolds.
  
  
  METHODS
  MDSCs were harvested from mice hind leg muscles using the preplate technique and stably transfected with a plasmid to express the LacZ reporter gene. Fifty different preparations of SIS cultured with MDSCs (MDSC/SIS) or SIS alone were incubated at 37 degrees C for 1, 4, and 8 weeks and also were mounted in a bath to measure the isometric contractions.
  
  
  RESULTS
  LacZ and Masson-trichrome staining revealed MDSCs could migrate into and distribute throughout the SIS and form myotubes. In MDSC/SIS, spontaneous contractile activities were noted in the 4-week (five of six specimens) and 8-week (eight of eight specimens) cultures, but not in 1-week cultures (n = 11). All SIS control groups after 1 (n = 11), 4 (n = 6), and 8 (n = 8) weeks of incubation did not show any activity. In most of the 4-week, and all of the 8-week, MDSC/SIS cultures, the frequency and amplitude of spontaneous contractile activities were decreased by succinylcholine 10 microM and 20 microM. Electrical field stimulation, carbachol, and KCl did not alter the frequency, amplitude, or pattern of spontaneous contractile activities in MDSC/SIS. Spontaneous contractile activities were blocked by Ca(32+)-free Krebs solution with ethyleneglycoltetraacetic acid 200 microM and distilled water.
  
  
  CONCLUSIONS
  MDSCs could be incorporated into SIS-forming myotubes capable of contracting. The contractile activity of this three-dimensional construct is Ca(2+) dependent and is modulated by nicotinic receptors. MDSC seeding of an acellular matrix may become a functional sling to reengineer the deficient sphincter or as contractile bladder augmentation.
authors.affiliation: Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Shing-Hwa
authors.initials: SH
authors.lastname: Lu
authors.forename: Tracy W
authors.initials: TW
authors.lastname: Cannon
authors.forename: Christopher
authors.initials: C
authors.lastname: Chermanski
authors.forename: Ryan
authors.initials: R
authors.lastname: Pruchnic
authors.forename: George
authors.initials: G
authors.lastname: Somogyi
authors.forename: Michael
authors.initials: M
authors.lastname: Sacks
authors.forename: William C
authors.initials: WC
authors.lastname: de Groat
authors.forename: Johnny
authors.initials: J
authors.lastname: Huard
authors.forename: Michael B
authors.initials: MB
authors.lastname: Chancellor
mesh_terms: D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000502:physiology*; D002469:Cell Separation; D002478:Cells, Cultured; D002642:Chick Embryo; D004305:Dose-Response Relationship, Drug; D006614:Hindlimb; D007413:Intestinal Mucosa / Q000378:metabolism; D007421:Intestine, Small / Q000378:metabolism; D051379:Mice; D018101:Mice, Inbred mdx; D039902:Multipotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal; D018733:Nicotinic Antagonists / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000502:physiology*; D013390:Succinylcholine / Q000494:pharmacology
chemical_list: D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D013390:Succinylcholine; D002118:Calcium