Yield optimisation and molecular characterisation of uncultured CD271+ mesenchymal stem cells in the reamer irrigator aspirator waste bag

Abstract

Bone reconstruction requires the use of autografts from patients' iliac crest (IC); for large-volume defects bone void fillers and autologous mesenchymal stem cells (MSCs) are often added. The Reamer/Irrigator/Aspirator (RIA) device provides the means of harvesting large amounts of autograft and additionally yields a waste bag containing MSCs, which is currently discarded. The aim of this study was to enumerate and characterise native MSCs from RIA waste bag and compare them to 'gold-standard' donormatched MSCs from IC bone marrow (BM). IC-BM from age matched trauma patients was used as control.In RIA waste bags the median MSC yield established using a colony-forming fibroblast assay was 314333 (range 5 x 10 4 -1.4 x 10 6 ), equivalent to approximately one litre of IC-BM aspirate. CD271+ cells were present at high levels in RIA waste bags, had MSC surface phenotype (CD90 + CD73 + CD105 + CD34 -CD61 -CD19 -CD31 -CD33 -) and expressed genes associated with multipotentiality, osteogenesis, adipogenesis and angiogenic support. RIA-CD271 + MSCs were transcriptionally similar to donormatched IC-CD271 + MSCs (76 % transcripts); with the majority of bone-related and Wnt pathway molecules being expressed at comparable levels. Lower-level expression of MCAM/CD146 and 5/13 hypoxia-related molecules was found in RIA-CD271 + MSCs, potentially reflecting their native residence in a more hypoxic environment of the endosteum and bone cortex.These data suggest that long bones contain very large numbers of MSCs, transcriptionally-similar to IC-BM MSCs; they can be procured by reaming using the RIA device and used, following concentration, as autologous and potentially allogeneic bone repair therapy.

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

“…MSCs identified by negative to low expression of CD45 and positive expression of CD271 (CD45 −/low CD271 + ) [16,32,34], as well as cells with positive expression of CD45 and low expression of CD271 (CD45 + CD271 low ) were examined in detail. Expression of a range of cell surface markers was examined by incubation with monoclonal antibodies; CD3, CD14, CD19, CD31, CD34, CD73, CD90, CD105, CD146, CD235a, mesenchymal stem cell antigen 1 (MSCA-1) and sushi domain containing 2 (SUSD2) [23,38,39] (Table 1).…”

“…TaqMan gene expression assays were then used according to the manufacturer’s instructions to measure transcript expression of genes important for bone regeneration. These were bone morphogenic protein 2 (BMP-2) an essential mediator of fracture healing [42], vascular endothelial growth factor C (VEGFC) an important stimulator of angiogenesis [43], Collagen type I alpha 2 (COL1A2) and osteonectin (SPARC) two factors involved in matrix deposition and mineralisation [34] and Stromal derived factor 1 (CXCL12) a chemokine capable of influencing chemotaxis of a wide range of cell types including MSCs [44,45]. Transcript expression was measured relative to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) in three matched samples, Pre and Post enrichment, from each tissue.…”

SOST, an LNGFR target, inhibits the osteogenic differentiation of rat ectomesenchymal stem cells

“…Previous reports have shown that LNGFR is involved in cell death and axonal degeneration in response to injury . Increasing numbers of studies have demonstrated that LNGFR can serve as a cell surface marker for mesenchymal stem cells (MSCs) and that LNGFR‐positive MSCs present increased potential for osteogenesis . In this study, EMSCs were isolated from embryonic facial processes and sorted to obtain LNGFR + EMSCs and LNGFR − EMSCs.…”

“…12 Increasing numbers of studies have demonstrated that LNGFR can serve as a cell surface marker for mesenchymal stem cells (MSCs) and that LNGFR-positive MSCs present increased potential for osteogenesis. [13][14][15][16][17][18] In this study, EMSCs were isolated from embryonic facial processes and sorted to obtain LNGFR + EMSCs and LNGFR − EMSCs. We further demonstrated that LNGFR + EMSCs had a higher osteogenic capacity and lower SOST levels compared with LNGFR − EMSCs and that SOST negatively regulated the osteogenic differentiation of EMSCs.…”

Bone marrow-derived mesenchymal stem cells migrate to healthy and damaged salivary glands following stem cell infusion

“…30 CD61, or integrin beta-3, is believed to be negative in MSCs. 31 CD90 (thymocyte differentiation antigen-1,Thy-1) is a positive marker recommended by the International Society for Cellular Therapy position statement. 24 CD106 (vascular cell adhesion molecule 1, VCAM-1) is expressed on activated endothelial cells, and it facilitates adhesion of haematopoietic cells to endothelial cells and subsequently their migration into tissue.…”