Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration

Abstract

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; 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.

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.

Fig 1. CD271-based enrichment of MSCs from BM aspirates.

Comparison of the morphology (x10 magnification) and population doubling time between PA-MSC (open bars) and CD271-MSC isolated using the QuadraMACS system (closed bars, n = 5); Bar charts show means + SEM (A). Immunophenotypic analysis of PA-MSC (open bars n = 5) and CD271-MSC (closed bars) from passage 1 to passage 3. (Lin corresponds to a cocktail of CD14, CD34, CD45 and CD19) (B). Representative dot plots generated from a single BM aspirate showing the CD45^−/lowCD73⁺ population (box) (C), the CD45^−/lowCD271⁺ population (large box) and the CD45⁺CD271^low population (small box) (D). The relationship between the proportion of CD45^−/lowCD73⁺ and CD45^−/lowCD271⁺ cells (23 separate BM aspirates collected from 12 donors) (E). The proportion of CD45^−/lowCD73⁺ (F) and CD45^−/lowCD73⁺CD271⁺ (G) cells, pre and post CliniMACS enrichment with representative dot plots below (n = 3). The proportion of total live cells (H) and CD45⁺ leukocytes (I) pre and post CliniMACS enrichment. The proportion of CD45⁺CD271^low ‘passenger’ cells (J), pre and post enrichment and a detailed analysis of ‘passenger cell’ phenotype (K). (Data F-K, n = 3)

Fig 2. CD271-based enrichment of MSCs from discarded femoral heads.

The proportion of CD45^−/lowCD73⁺ (A) and CD45^−/lowCD73⁺CD271⁺ (B) cells, from enzymatically digested femoral heads pre and post CliniMACS enrichment with representative dot plots below. The proportion of CFU-Fs pre and post CliniMACS enrichment with representative images of CFU-F dishes below (C). The proportion of CD45⁺CD271^low ‘passenger’ cells (D), CD45⁺ leukocytes (E) and total live cells (F) pre and post CliniMACS enrichment. (All data n = 3).

Fig 3. CD271-based enrichment of MSCs from RIA waste fluid.

Inter-operative images of the RIA device showing the main body of the device (left panel), the reamer head passing through the intramedullary canal (middle panel) and the waste fluid collection bag (right panel) (A). The proportions of CD45^−/lowCD73⁺ (B) and CD45^−/lowCD73⁺CD271⁺ cells (C) in RIA waste fluid pre and post CliniMACS enrichment, with representative dot plots shown below. The proportion of CFU-Fs pre and post CliniMACS enrichment and representative CFU-F dishes below (D). The proportion of CD45⁺CD271^low ‘passenger’ cells (E), CD45⁺ leukocytes (F) and total live cells (G) pre and post CliniMACS enrichment. (All data n = 3).

Fig 4. Functional analysis, phenotypic profile and transcripts expression of CD271 selected MSCs from BM, FH and RIA.

Differentiation potential of PA-MSCs and CD271-MSCs. Osteogenesis, adipogenesis and chondrogenesis was measured on day-21 post-induction by staining with alkaline phosphatase/von Kossa, Oil Red O and Alcian Blue, respectively in cells selected from BM (A), FH (B) and RIA (C), all size bars represent 500μm. Phenotypic analysis of the CD45^−/lowCD271⁺ population observed in BM (D), FH (E) and RIA (F). Analysis of the total expression of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts relative to HPRT in BM (G), FH (H) and RIA (I) pre (black bars) and post (grey bars) CD271 enrichment. (All data n = 3).