Mesenchymal content of fresh bone marrow: a proposed quality control method for cell therapy

Abstract

Summary The scarcity of mesenchymal stem cells (MSC) in bone marrow (BM) has justified their ex vivo expansion before therapeutic use, but a method to evaluate the quality of initial mesenchymal content and track the modifications induced by graft processing has not yet been proposed. The aim of this study was to establish such a procedure. Flow cytometric and functional assay methods were modified to count CD45− CD14−/CD73+ subsets containing all MSC and used them to study BM from spongy bone (SB) and iliac crest aspirate (ICA). These methods detected the target subsets in all BM suspensions derived from SB (n = 154) and ICA, (n = 44) with a satisfactory correlation between immuno‐phenotyping and functional tests by low‐density plating. We noted a higher overall MSC frequency in SB cell suspensions but a lower plating efficiency of CD45− CD14−/CD73+ SB cells under standard culture conditions. We propose a cell quality control on un‐manipulated BM cell suspensions to quantify the mesenchymal compartment with regard to varying donor factors, such as age and sampling site, that influence expansion and define a therapeutic threshold value. Furthermore, we were able to confirm differences in plating efficiency and proliferative capacity between two BM origins.

Cell-Surface Expression of Neuron-Glial Antigen 2 (NG2) and Melanoma Cell Adhesion Molecule (CD146) in Heterogeneous Cultures of Marrow-Derived Mesenchymal Stem Cells

“…MSCs must be expanded ex vivo for most clinical applications 49 due to their rarity in tissues. 50 Our sort strategy may aid in isolating MSCs with the ability to expand sufficient quantities of cells ex vivo for MSC therapies. Moreover, our results may enable manufacturers to monitor and enrich MSC populations during ex vivo expansion as a means to standardize the composition of MSC therapies.…”

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

“…Because MSCs are very rare in BM aspirates [16–19], the isolation of pure uncultured BM MSCs in the ‘research-scale’ settings is best achieved by cell sorting with a combination of positive and negative markers [17,20–23]. Amongst a number of positive markers proposed in the past [24–26], CD271 offers unique selectivity, defined as the least cross-reactivity with contaminating haematopoietic-lineage cells [7,17,23,27,28].…”

Processing Volumes and Therapeutic Cellular Doses of Point of Care Bone Marrow Concentrates

“…This protocol made it possible to adjust BMC volumes to desirable final values in order to fit defined anatomic spaces while maintaining MSC doses with previously reported therapeutic effects. On the other hand, it is really surprising that while an overwhelming part of the cells within BMC belong to hemopoetic lineages and MSC represent approximately only a 0.003 % of TNC [24] , those blood cells are usually underestimated in terms of its medicinal potential. In this regard neutrophils, which are BMC main cellular component representing more than 70% of living cells, could also be directly involved in BMC´s observed therapeutic effects.…”

“…Quantification of Mesenchymal stromal cells (MSC) was performed by means of Fibroblastic Colony Forming units (CFU-F) assay in all cases as previously reported [24] . Briefly, CFU-F were determined by platting 5 × 10 4 living total nucleated cells (TNC) per cm 2 in 6 well dishes per triplicate.…”