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

Abstract

IntroductionCurrent 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 MethodsMSCs 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.ResultsUncultured 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.DiscussionA 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.

Mesenchymal Stem/Stromal Cells in Regenerative Medicine: Can Preconditioning Strategies Improve Therapeutic Efficacy

“…However, with the limitations of a potentially decreasing purity during sub-culturing and/or producing ‘culture artifacts', as interplay of subpopulations might be relevant for their function, the CD271+ MSC subpopulation is abundant in intramedullary cavities of the long bones, and CD271+ sorted BM-MSCs featured greater osteogenic differentiation potential compared to non-sorted BM-MSCs [32,51]. Meanwhile, first steps have been taken towards clinical-grade GMP production of CD271+ sorted BM-MSCs for bone regeneration [52]. Amongst placenta-derived MSCs, a greater osteogenic differentiation potential could be assigned to the CD146+ subpopulation, whereas, after sorting, CD146- MSCs could not form mineralized extracellular matrix, an indicator for functional osteogenic differentiation [53].…”

Isolation and Characterization of Human Mesenchymal Stromal Cell Subpopulations: Comparison of Bone Marrow and Adipose Tissue

“…Quirici et al [36] obtained a cell purity of 90.5±3.5% for BM CD271+ cells after the purification of CD45α-glycophorin A -BM cells. In other studies, the percentage of CD271 was evaluated by flow cytometry after CD45gating of the selected population [50]. Higher isolation purity could be also achieved by a second run of purification.…”

Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells-An in vitro proof-of-concept study

“…In addition, it has been shown that the CD271 pos population represents primary stem cells with MSC phenotype fulfilling stringent functional stem cell criteria in vivo (Ghazanfari et al, ). The cells have been shown to exhibit the essential trilineage potential and were phenotypically identical to plastic adherence‐selected MSCs following expansion culture (Colosimo et al, ; Cuthbert et al, ). In contrast to the results of Rozemueller et al, who demonstrated an enrichment of CFU‐Fs for flow‐sorted ovine W4A5 pos and MSCA1 pos cells, it was not possible to enrich clonogenic MSCs by using these markers for separation in the present study.…”