Nonadherent culture method downregulates stem cell antigen-1 expression in mouse bone marrow mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) are primarily isolated by their adherence to plastic and their in vitro growth characteristics. Expansion of these cells from an adherent culture is the only method to obtain a sufficient number of cells for use in clinical practice and research. However, little is known with regard to the effect of adherence to plastic on the phenotype of the cells. In the present study, bone marrow CD45^-CD31^-CD44^- stem cell antigen (Sca)-1⁺ MSCs were sorted by flow cytometry and expanded in adherent cultures. The expression levels of the adhesion molecule, Sca-1, in the adherent cultures were compared with those from nonadherent cultures at different time points. The flow cytometry results indicated that the expression levels of Sca-1 decreased in the MSCs in the nonadherent cultures grown in ultra-low-adherent plates. Furthermore, the result was confirmed by quantitative polymerase chain reaction at the same time points. Therefore, the results demonstrated that the loss of plastic adherence downregulated the expression of Sca-1. The observations may provide novel insights into the molecular mechanisms underlying plastic adherent culture.

Keywords: adherent culture; mesenchymal stem cells; nonadherent culture; phenotype; stem cell antigen-1.

Figure 1.

Fluorescence-activated cell sorting (FACS) isolation of mouse bone marrow mesenchymal stem cells (BM MSCs). FACS profile showing the gating strategy for the sorting of BM MSCs (CD45⁻LIN⁻CD31⁻CD44⁻Sca-1⁺). The CD31⁻CD45⁻LIN⁻ cells were first gated within the live cells (PI⁻), after which the CD44⁻Sca-1⁺ cells were gated. Sca, stem cell antigen; LIN, lineage markers; PI, propidium iodide.

Figure 2.

Experimental procedure and morphology of nonadherent and adherent cultured cells. (A) MSCs were sorted by FACS and expanded to eight passages by culturing in plastic. Subsequently, the MSCs were seeded into ultra-low-attachment culture plates and resuspended in the medium (nonadherent culture). As a control, the same number of MSCs were seeded in tissue culture plates and attached to the bottom of the plate (adherent culture). Nonadherent and adherent cultured cells obtained after 72 h were reseeded into tissue culture plates and incubated for 5 days (5 days adherent culture). FACS and quantitative polymerase chain reaction were performed at 24 h, 72 h and 5 days. (B) Morphology of the MSCs. MSCs were plated in the tissue culture plates and cultured for (a) 24 h, (b) 72 h and (c) 5 days. All the MSCs in images a-c were attached to the bottom of the plastic plate. MSCs were plated in ultra-low-attachment tissue culture plates and cultured for (d) 24 h and (e) 72 h. All the MSCs from stages d and e were suspended in the medium. (f) MSCs cultured for 72 h in the nonadherent culture were reseeded into tissue culture plates and cultured for 5 days. The cells were attached to the bottom of the plastic plate. Scale bar, 100 µm. FACS, fluorescence-activated cell sorting; BM MSCs, bone marrow mesenchymal stem cells.

Figure 3.

Flow cytometry analysis of the expression of the Sca-1 surface marker. (A, C and E) Graphs represent Sca-1 expression on the adherent cultured cells at 24 h, 72 h and 5 days, respectively. (B, D and F) Graphs represent Sca-1 expression on the nonadherent cultured cells at 24 h, 72 h and 5 days, respectively. Expression of the Sca-1 surface marker was analyzed by fluorescence-activated cell sorting, and the expression levels were compared between the adherent and nonadherent cultured cells. Three independent experiments were performed with the cells between passages 9 and 11, which all showed similar Sca-1 expression. (G) Statistical histogram shows the differences in Sca-1 expression levels at 24 h (P>0.05), 72 h (*P<0.05) and 5 days (P>0.05) between the nonadherent and adherent cultured cells. FITC, fluorescein isothiocyanate; Ad, adherent; Nonad; nonadherent; Sca, stem cell antigen.

Figure 4.

Quantitative polymerase chain reaction (qPCR) analysis. Expression of the Sca-1 gene was determined by qPCR. Adherent and nonadherent cultured cells between passages 9 and 11 were used in the experiment. The data are the average of three independent experiments. (A) Sca-1 expression in the nonadherent cultured cells at 24 h, as compared with the adherent cultured cells at 24 h (P>0.05). (B) Sca-1 expression in the nonadherent cultured cells at 72 h compared with the adherent cultured cells at 72 h (**P<0.01). (C) Sca-1 expression in the nonadherent cultured cells at 5 days, as compared with the adherent cultured cells at 5 days (*P<0.05). These experiments demonstrated that the mRNA expression levels of Sca-1 in the mesenchymal stem cells were downregulated after 72 h in the nonadherent culture; however, Sca-1 mRNA expression recovered following culture for 5 days in the adherent culture conditions. Sca, stem cell antigen; Ad, adherent; Nonad, nonadherent.