title: Neuronal hypoxia in vitro: investigation of therapeutic principles of HUCB-MNC and CD133+ stem cells.
abstract: BACKGROUND
  The therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood.
  
  
  METHODS
  A human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC), a stem cell enriched fraction (CD133+, 97.38% CD133-positive cells) and a stem cell depleted fraction (CD133-, 0.06% CD133-positive cells) of HUCB-MNC by either direct or indirect co-cultivation with post-hypoxic neuronal cells (differentiated SH-SY5Y). Over three days, development of apoptosis and necrosis of neuronal cells, chemotaxis of MNC and production of chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9) and growth factors (G-CSF, GM-CSF, VEGF, bFGF) were analyzed using fluorescence microscopy, FACS and cytometric bead array.
  
  
  RESULTS
  tMNC, CD133+ and surprisingly CD133- reduced neuronal apoptosis in direct co-cultivations significantly to levels in the range of normoxic controls (7% +/- 3%). Untreated post-hypoxic control cultures showed apoptosis rates of 85% +/- 11%. tMNC actively migrated towards injured neuronal cells. Both co-cultivation types using tMNC or CD133- reduced apoptosis comparably. CD133- produced high concentrations of CCL3 and neuroprotective G-CSF within indirect co-cultures. Soluble factors produced by CD133+ cells were not detectable in direct co-cultures.
  
  
  CONCLUSIONS
  Our data show that heterogeneous tMNC and even CD133-depleted fractions have the capability not only to reduce apoptosis in neuronal cells but also to trigger the retaining of neuronal phenotypes.
authors.affiliation: Fraunhofer-Institute for Cell Therapy and Immunology, Perlickstrasse 1, 04103 Leipzig, Germany. doreen.reich@izi.fraunhofer.de
authors.forename: Doreen M
authors.initials: DM
authors.lastname: Reich
authors.forename: Susann
authors.initials: S
authors.lastname: Hau
authors.forename: Tobias
authors.initials: T
authors.lastname: Stahl
authors.forename: Markus
authors.initials: M
authors.lastname: Scholz
authors.forename: Wilfried
authors.initials: W
authors.lastname: Naumann
authors.forename: Frank
authors.initials: F
authors.lastname: Emmrich
authors.forename: Johannes
authors.initials: J
authors.lastname: Boltze
authors.forename: Manja
authors.initials: M
authors.lastname: Kamprad
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000032:analysis*; D017209:Apoptosis; D002454:Cell Differentiation; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018925:Chemokines / Q000032:analysis / Q000096:biosynthesis; D018920:Coculture Techniques / Q000379:methods; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D006023:Glycoproteins / Q000032:analysis*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009447:Neuroblastoma / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D010455:Peptides / Q000032:analysis*
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018925:Chemokines; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; C000606023:PROM1 protein, human; D010455:Peptides