Standardized quantification of human bone marrow mesenchymal stromal cells based on a flow cytometric assay

Abstract

Conclusion The mesenchymal stromal cell (MSC) content is a major quality determinant of human bone marrow aspirates (BMA). It depends on the aspiration site, contamination with peripheral blood, as well as the aspiration procedure, and it negatively correlates with donor age. The common way of quantifying MSCs is based on the cultivation of cells using the colony forming unit– fibroblast (CFU-F) assay, which is time consuming and highly variable. Cuthbert et al. described a close linear relationship between the number of CFU-F colonies counted manually after 14 days of culture and the number of CD271bright cells per mL of BMA1. The aim of this work was the optimization of a fast and reproducible flow cytometry–based MSC quantification method enabling prospective quality assessment of BMA. In previous experiments we have shown that only CD271bright cells, which also express MSCA-1, give rise to CFU-F. MSCs were isolated from 2×107 bone marrow mononuclear cells (BM MNC) using CD271 (LNGFR)-APC and Anti-APC MicroBeads. Samples of all fractions were labeled with CD45-FITC. PI fluorescence and light scatter signals were used for gating live cells. The positive fraction contained CD271+ cells with a purity of about 86% (Mean: 83±4.1%; n = 4). This population contained about 73% CD271CD45 cells (A, orange fraction) and about 13% CD271CD45 cells (A, red fraction). Further separation of CD271 and CD271 cells was achieved by flow sorting. The different fractions were cultivated and CFU-F numbers were counted after 14 days. The numbers of CFU-F were about 40-fold higher when MSCs were cultivated from the CD271/ CD45 fraction compared to MSCs obtained by plastic adherence (PA). No CFU-Fs were detected in the CD271– and CD271CD45 cell fractions (A). Counterstaining of isolated CD271+ cells revealed a 100% co-expression of MSCA-1 in CD271CD45 cells (B). CD271CD45 MSCs met ISCT criteria2 after culture expansion (3 passages) with respect to MSCspecific marker expression (C) and their differentiation potential towards osteocytes, adipocytes, and chondrocytes (D, left to right)