Examining the Feasibility of Clinical Grade CD271(+) Enrichment of Mesenchymal Stromal Cells for Bone Regeneration

作者：Cuthbert Richard J^*; Giannoudis Peter V; Wang Xiao N; Nicholson Lindsay; Pawson David; Lubenko Anatole; Tan Hiang B; Dickinson Anne; McGonagle Dennis; Jones Elena

来源：PLos One, 2015, 10(3): UNSP e0117855.

DOI：10.1371/journal.pone.0117855

Introduction
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.
Materials and Methods
MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzy-matically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45(-/low) CD73(+) 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(-/ low) CD271(+) 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.
Discussion
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.

• 出版日期2015-3-11