title: Two steps to functional mesenchymal stromal cells for clinical application.
abstract: BACKGROUND
  Ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) is a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. Even large volumes of starting material and extended culture periods, however, do not necessarily produce 2 x 10(6) MSCs per kg per adult patient. A new two-step procedure has been devised to propagate more than 1 x 10(8) MSCs from small marrow volumes within fewer than 4 weeks.
  
  
  METHODS
  The influence of log fold decreased MSC seeding (2500, 250, 25, 2.5/cm(2)) on clinical-scale expansion, MSC phenotype, and immunomodulatory function combined with multiplex cytokine display was analyzed. Maintenance of MSC characteristics was tested in fibroblast colony-forming unit and differentiation assays.
  
  
  RESULTS
  Reduced seeding density boosted MSC propagation. Low-density expanded MSCs were CD29+, CD73+, CD90+, CD105+, CD14-, CD34-, CD45-, HLA-DR-; retained their differentiation potential; and inhibited lymphocyte proliferation. This was accompanied by deregulated cytokine production. Seeding 0.7 x 10(6) to 1 x 10(6) MSCs derived from a 10- to 13-day primary culture at a low density of 28 to 40 per cm(2) permitted propagation of 1.5 x 10(8) to 3.7 x 10(8) functional MSCs within a 13- to 15-day secondary expansion step.
  
  
  CONCLUSIONS
  Primary seeding of only 10-mL marrow aspirates on approximately 0.2-m(2) culture area (Step 1) followed by expansion on 2.5 m(2) (Step 2) is sufficient to consistently generate at least 1.5 x 10(8) MSCs in fetal bovine serum-supplemented medium within less than 4 weeks. The efficiency of this two-step procedure for clinical-scale MSC propagation may facilitate rational clinical testing of MSC-based therapies.
authors.affiliation: Department of Internal Medicine, Division of Hematology and Stem Cell Transplantation, Medical University, Auenbrugger Platz 38, A-8036 Graz, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Bartmann
authors.forename: Eva
authors.initials: E
authors.lastname: Rohde
authors.forename: Katharina
authors.initials: K
authors.lastname: Schallmoser
authors.forename: Peter
authors.initials: P
authors.lastname: Pürstner
authors.forename: Gerhard
authors.initials: G
authors.lastname: Lanzer
authors.forename: Werner
authors.initials: W
authors.lastname: Linkesch
authors.forename: Dirk
authors.initials: D
authors.lastname: Strunk
mesh_terms: D000328:Adult; D000368:Aged; D001854:Bone Marrow Cells / Q000166:cytology; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008875:Middle Aged; D017154:Stromal Cells / Q000166:cytology*