title: Human bone marrow stromal cell confluence: effects on cell characteristics and methods of assessment.
abstract: OBJECTIVE
  Ex vivo expansion and serial passage of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) is required to obtain sufficient quantities for clinical therapy. The BMSC confluence criteria used to determine passage and harvest timing vary widely, and the impact of confluence on BMSC properties remains controversial. The effects of confluence on BMSC properties were studied and confluence-associated markers were identified.
  
  
  METHODS
  BMSC characteristics were analyzed as they grew from 50% to 100% confluence, including viability, population doubling time, apoptosis, colony formation, immunosuppression, surface marker expression, global gene expression and microRNA expression. In addition, culture supernatant protein, glucose, lactate and pH levels were analyzed.
  
  
  RESULTS
  Confluence-dependent changes were detected in the expression of several cell surface markers: 39 culture supernatant proteins, 26 microRNAs and 2078 genes. Many of these surface markers, proteins, microRNAs and genes have been reported to be important in BMSC function. The pigment epithelium-derived factor/vascular endothelial growth factor ratio increased with confluence, but 80% and 100% confluent BMSCs demonstrated a similar level of immunosuppression of mixed lymphocyte reactions. In addition, changes in lactate and glucose levels correlated with BMSC density.
  
  
  CONCLUSIONS
  BMSC characteristics change as confluence increases. 100% confluent BMSCs may have compromised pro-angiogenesis properties but may retain their immunomodulatory properties. Supernatant lactate and glucose levels can be used to estimate confluence and ensure consistency in passage and harvest timing. Flow cytometry or microRNA expression can be used to confirm that the BMSCs have been harvested at the appropriate confluence.
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Jiaqiang
authors.initials: J
authors.lastname: Ren
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Huan
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Tran
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Civini
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Jin
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Luciano
authors.initials: L
authors.lastname: Castiello
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Feng
authors.affiliation: Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Kuznetsov
authors.affiliation: Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Pamela G
authors.initials: PG
authors.lastname: Robey
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Marianna
authors.initials: M
authors.lastname: Sabatino
authors.affiliation: Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: dstroncek@cc.nih.gov.
authors.forename: David F
authors.initials: DF
authors.lastname: Stroncek
mesh_terms: D017209:Apoptosis / Q000502:physiology; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000502:physiology*; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D002478:Cells, Cultured; D005136:Eye Proteins / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D020869:Gene Expression Profiling; D005947:Glucose / Q000378:metabolism; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics; D009414:Nerve Growth Factors / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D015415:Biomarkers; D005136:Eye Proteins; D005947:Glucose; D019344:Lactic Acid; D008565:Membrane Proteins; D035683:MicroRNAs; D009414:Nerve Growth Factors; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor
keywords: bone marrow stromal cells; cellular therapy; confluence; gene expression profiling; microRNA