Nanoparticle labeling of bone marrow-derived rat mesenchymal stem cells: their use in differentiation and tracking

Abstract

Mesenchymal stem cells (MSCs) are promising candidates for cellular therapies due to their ability to migrate to damaged tissue without inducing immune reaction. Many techniques have been developed to trace MSCs and their differentiation efficacy; however, all of these methods have limitations. Conjugated polymer based water-dispersible nanoparticles (CPN) represent a new class of probes because they offer high brightness, improved photostability, high fluorescent quantum yield, and noncytotoxicity comparing to conventional dyes and quantum dots. We aimed to use this tool for tracing MSCs' fate in vitro and in vivo. MSC marker expression, survival, and differentiation capacity were assessed upon CPN treatment. Our results showed that after CPN labeling, MSC markers did not change and significant number of cells were found to be viable as revealed by MTT. Fluorescent signals were retained for 3 weeks after they were differentiated into osteocytes, adipocytes, and chondrocytes in vitro. We also showed that the labeled MSCs migrated to the site of injury and retained their labels in an in vivo liver regeneration model. The utilization of nanoparticle could be a promising tool for the tracking of MSCs in vivo and in vitro and therefore can be a useful tool to understand differentiation and homing mechanisms of MSCs.

Figure 1

Characterization and labelling efficiency of 24 h CPN treated and nontreated MSCs. (a) After CPN-labeling MSCs (left panel) were positive for mesenchymal stem cell markers (CD90, CD29, and CD71) and negative for hematopoietic stem cell markers (CD34 and CD45) same as nontreated MSCs (right panel). β-Actin was used for loading control. (b) CPN-MSCs were visualized by fluorescence microscopy by using FITC filters. DAPI staining was performed to visualize cellular DNA. Magnification bar: 100 μm.

Figure 2

Effects of 24 h CPN labeling on MSCs' (a) cellular metabolic activity shown by MTT assay and (b) cellular toxicity shown by LDH assay (^* P < 0.005).

Figure 3

Differentiation of CPN-labeled MSCs into ((a), (b)) osteogenic lineage, ((c), (d)) adipogenic lineage, and ((e), (f)) chondrogenic lineage after culturing with induction medium. (a) Visualization of osteogenic lineage by Alizarin Red staining; (c) adipogenic lineage by Oil Red O staining; and (e) chondrogenic lineage by Alcian Blue staining under bright-field microscopy. ((b), (d), and (f)) Visualization of CPN labeling shown by using FITC filters since CPN had a similar emission and excitation wavelength to FITC. Red arrows denote some of the positive staining for the presence of CPNs after differentiation. Magnification bar: 20 μm.

Figure 4

In vivo tracking of CPN-MSCs. Paraffin embedded liver tissues obtained from rats that were injected with either CPN-labeled ((a)–(f)) or nonlabeled MSCs ((g)–(i)) from tail vein followed by PH ((a)–(c) and (g)–(i)) and SH ((d)–(f)). White arrows denote injected CPN-labelled MSCs under FITC filter. Magnification bar: 10 μm.