title: Culture bag systems for clinical applications of adult human neural crest-derived stem cells.
abstract: BACKGROUND
  Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags.
  
  
  METHODS
  We cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs.
  
  
  RESULTS
  When cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing β-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling.
  
  
  CONCLUSIONS
  Here, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine.
authors.forename: Johannes F W
authors.initials: JF
authors.lastname: Greiner
authors.forename: Lena-Marie
authors.initials: LM
authors.lastname: Grunwald
authors.forename: Janine
authors.initials: J
authors.lastname: Müller
authors.forename: Holger
authors.initials: H
authors.lastname: Sudhoff
authors.forename: Darius
authors.initials: D
authors.lastname: Widera
authors.forename: Christian
authors.initials: C
authors.lastname: Kaltschmidt
authors.forename: Barbara
authors.initials: B
authors.lastname: Kaltschmidt
mesh_terms: D000328:Adult; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D009432:Neural Crest / Q000166:cytology*; D058953:Neural Stem Cells / Q000166:cytology*; D014162:Transfection