Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow

Abstract

Introduction: Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs.

Methods: Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13(high) CD105(+) CD45(-) cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined.

Results: Our results show that the CD13(high) CD105(+) CD45(-) immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR.

Conclusions: BM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13(high) CD105(+) and CD45(-) immunophenotypic profile. Furthermore, in vitro expansion of these cells is associated with significant changes in the immunophenotypic profile of MSCs.

Fig. 1

Representative bivariate dot plots and single parameter histograms illustrating the gating strategy and the ex vivo immunophenotypic characteristics of bone marrow CD13^high CD105⁺ CD45^– cells. a–h Gating strategy used for the identification of these bone marrow cells based on their unique CD13^high CD105⁺ CD45^– and high but heterogeneous light scatter features. i–t Immunophenotypic features of bone marrow CD13^high CD105⁺ CD45^– cells from a representative healthy donor. Baseline autofluorescence and expression levels for each protein are indicated in the histogram plot in gray and black, respectively, using the overlay histogram function of the Infinicyt software. SSC sideward light scatter, MFI median fluorescence intensity (arbitrary units scaled from 0 to 262,000)

Fig. 2

Growth kinetics (a) and immunophenotypic features (b–h) of mesenchymal stem cells (MSCs) obtained from BM mononuclear cells (MNCs) using a standard protocol for in vitro MSC expansion compared to the growth kinetics of FACS-sorted and in vitro cultured CD13^high CD105⁺ CD45^– BM cells (identified as CD13^high cells). a Bars and vertical lines correspond to median ± 95 % confidence interval values. b–h Notched boxes extend from the 25th to 75th percentile values; the lines in the middle and vertical lines correspond to median values and both the 10th and 90th percentiles, respectively. Normalized median fluorescence intensity (MFI) values are displayed after subtracting the background autofluorescence levels observed for each individual marker-associated fluorochrome channel (arbitrary units scaled from 0 to 262,000). NS not significant (P > 0.05)

Fig. 3

The functional features of mesenchymal stem cells (MSCs) observed in vitro after culture of Biocoll isolated mononuclear cells (MNCs) with conventional standard plastic adherence methods (left panels) versus FACS-purified and cultured CD13^high CD105⁺ CD45^– BM cells (right panels). Panels I a and I b show undifferentiated and unstained MSCs cultured for 24 days (passage 3). Panels II through V display cells which have been induced to differentiate in vitro towards the osteogenic (Panels II and III), adipogenic (Panels IV) and chondrogenic (Panels V) differentiation pathways. Osteogenic differentiation was assessed using alkaline phosphatase (Panel II) and alizarin red S (Panel III) stainings at days +8 and +11, respectively; adipogenesis was determined using the oil red O staining at day +8 (Panel IV), and chondrogenesis was evaluated by toluidine blue staining at day +27 (Panel V). For each staining technique in Panels II–IV, undifferentiated controls counterstained with hematoxylin are also displayed (Panels a and c)