High abundance of CD271+ multipotential stromal cells (MSCs) in intramedullary cavities of long bones

Abstract

Aspiration of iliac crest bone marrow (ICBM) remains the most frequent technique used in harvesting multipotential stromal cells (MSCs) for bone regeneration. Although this tissue type is easily accessed by a surgeon, it has a low frequency of MSCs, which is significant given the high cell numbers required for bone regeneration strategies. Lipoaspirates possess higher MSC frequencies, albeit cells with a differentiation profile less suited to orthopaedic interventions. Intra-medullary cavities of long bones have previously been shown to harbour MSCs in animals, however evaluation of their frequency, differentiation capacity and phenotype in humans had not previously been performed.Long bone fatty bone marrow (LBFBM) was collected prior to harvesting bone graft. Basic cellular compositions of donor-matched LBFBM and ICBM aspirates, including the numbers of CD34+ hematopoietic stem cells and CD31+ endothelial cells, were similar. MSCs were enumerated using colony-forming-unit-fibroblast assays and flow cytometry for the presence of a resident LBFBM CD45−/low CD271+ MSC population and revealed a trend for higher MSC numbers (average 5 fold, n = 6) per millilitre of LBFBM compared to donor-matched ICBM. Functional characteristics of resident MSCs, including their growth rates, differentiation potentials and surface phenotypes (CD73+CD105+CD90+) before and after culture-amplification, were similar. Enhanced numbers of MSCs could be recovered following brief enzymatic treatment of solid fragments of LBFBM.Our findings therefore reveal that the intramedullary cavity of the human femur is a depot of MSCs, which, although closely associated with fat, have a differentiation profile equivalent to ICBM. This anatomical site is frequently accessed by the orthopaedic/trauma surgeon and aspiration of the intramedullary cavity represents a ‘low-tech’ method of harvesting potentially large numbers of MSCs for regenerative therapies and research.This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism.

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

“…3b, CD271 + cells derived from both IC BM aspirate and RIA fluid were positive for classical MSC markers CD73 and CD90 (Dominici et al, 2006;VeyratMasson et al, 2007). The expression of CD105, another well-known MSC marker (Barry et al, 1999;Pittenger et al, 1999) was slightly lower compared to CD73 and CD90, as described previously (Martinez et al, 2007;Cox et al, 2012), but remained highly specific for CD271 + cells. Consistent with their non-haematopoietic nature, CD271 + cells from both sources did not express markers for haematopoietic progenitors (CD34), megakaryocytes/ platelets (CD61), B-cells (CD19), endothelial cells (CD31) and myeloid cells (CD33).…”

“…In other words, the presence of MSCs is inferred by analysing their progeny whereas their native characteristics remain unknown. We were the first group to use the CD271 + phenotype to characterise native MSCs in the IC BM (Jones et al, 2002; and in long bones (Cox et al, 2012), later confirmed independently (Buhring et al, 2007;Kuci et al, 2011;Tormin et al, 2011;Maijenburg et al, 2012;Rasini et al, 2013 …”

“…When BM is removed from human long bones manually (by scooping), it contains only 2-to 5-fold more MSCs, compared to IC BM (Cox et al, 2012). This suggests that significantly greater numbers of MSCs recovered by reaming were most likely derived from the endosteum, which is disrupted by reaming but left intact during manual collection.…”

“…For flow cytometry validation, the RIA fluid was centrifuged to minimise the volume, before isolating mononuclear cells (MNCs) using a Lymphoprep ® density gradient (Axis-Shield, Dundee, UK); CD271 + cells were subsequently enumerated as described previously (Cox et al, 2012).…”

“…3 a,b). In our previous studies, we used the CD45 + CD271 -cell population (haematopoietic lineage cells, HLCs) as non-MSC 'negative' controls Cox et al, 2012), the same strategy was used in the present study (Fig. 3a, left panels).…”