title: Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration.
abstract: BACKGROUND
  Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells.
  
  
  METHODS
  MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair.
  
  
  RESULTS
  Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs.
  
  
  CONCLUSIONS
  A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Cuthbert
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.
authors.forename: Peter V
authors.initials: PV
authors.lastname: Giannoudis
authors.affiliation: Institute of Cellular Medicine, University of Newcastle upon Tyne, Newcastle, United Kingdom.
authors.forename: Xiao N
authors.initials: XN
authors.lastname: Wang
authors.affiliation: Institute of Cellular Medicine, University of Newcastle upon Tyne, Newcastle, United Kingdom.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Nicholson
authors.affiliation: National Health Service Blood and Transplant, Leeds Blood Centre, Leeds, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Pawson
authors.affiliation: National Health Service Blood and Transplant, Leeds Blood Centre, Leeds, United Kingdom.
authors.forename: Anatole
authors.initials: A
authors.lastname: Lubenko
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.
authors.forename: Hiang B
authors.initials: HB
authors.lastname: Tan
authors.affiliation: Institute of Cellular Medicine, University of Newcastle upon Tyne, Newcastle, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Dickinson
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.
authors.forename: Dennis
authors.initials: D
authors.lastname: McGonagle
authors.affiliation: Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom.
authors.forename: Elena
authors.initials: E
authors.lastname: Jones
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001861:Bone Regeneration; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002648:Child; D002675:Child, Preschool; D005270:Femur Head / Q000166:cytology; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010641:Phenotype; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D055815:Young Adult
chemical_list: C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D017475:Receptors, Nerve Growth Factor