Identification of suitable labelling method for tracking of MSCs

As mentioned in the introduction, several labelling methods can be utilized for imaging MSCs. For the purpose of this study, the suitability of the following methods has been assessed for detecting MSC following their incorporation into mouse kidney rudiment chimeras: (i) labelling with the vital cell tracker, CFDA SE; (ii) QD labelling; (iii) lentiviral transduction with GFP, (iv) staining with a species-specific antibody which was used only in conjunction with human MSCs.

In the first instance, the suitability of two transient labelling methods was assessed, in order to avoid potential problems associated with genetic modification. To be effective, the labelling method would need to show high labelling efficiency and low cytotoxicity. Furthermore, as the concentration of non-genetically encoded fluorophores is reduced with each cell division (Schroeder 2008), it was important to find a labelling method that would enable detection after several days of culture. A comparison was made between two transient labelling methods, CFDA SE and QDs, in order to elucidate their suitability for long term tracking. D1 cells were labelled with 10 µM CFDA SE or 10 nM QDs according to manufacturer’s instructions.

Figure 3.4 Confirmation of multilineage differentiation potential of human primary MSCs following stimulation with inductive media. (a) Stimulated with adipogenic inductive medium human MSCs after 3 cycles of adipogenic induction and subsequent week of culture in maintenance medium accumulated lipid vacuoles that stained positively with Oil Red. (b) Oil Red staining in the absence of stimulation: no lipid vacuoles were present in human MSCs cultured in maintenance medium only for the same period of time. (c) Alizarin Red staining indicated the presence of extracellular calcium deposits in human MSC cultures stimulated with osteogenic inductive medium after 14 days of culture. (d) Alizarin Red staining in control MSCs maintained in standard culture medium showed that no calcium deposits were present after 14 days of culture. c d 100µm 100µm 200µm 200µm b a

No visible difference in labelling efficiency was detected when the cells were analyzed directly after staining. All cells were stained following labelling with either CFDA SE (Figure 3.5a and b) or QDs (Figure 3.5e and f). Since CFDA SE interacts with intracellular molecules upon labelling, a diffuse cytoplasmic staining was observed (Figure 3.5a and b), whereas QDs showed a patchy pattern as nanoparticles tend to be unequally distributed in the cells (Figure 3.5e and f). After 5 days of standard culture no obvious cytotoxic effect was observed in any of the conditions. However, only the cells incubated with QDs remained labelled. As shown in Figure 3.5e-h QD staining remained intense, although in comparison to day 0 notably fewer cells were labelled with QDs. CFDA SE, on the other hand, was not detectable on the 5th day following labelling (Figure 3.5c and d).

Stable transduction or transfection with GFP is an efficient labelling method that should not lead to loss of signal over a period of time as the cells constitutively express the fluorescent protein that is used to detect them (Schroeder 2008). In this study, a lentivirus encoding enhanced GFP under control of the spleen focus-forming virus (SFFV) promoter has been used to induce expression of GFP in mouse MSCs. D1 cells were incubated for 24h with lentiviral particles carrying GFP (obtained from Sokratis Theocharatos, University of Liverpool). On the 3rd day following transduction, the cells were analyzed for GFP expression. No visible signs of cytotoxicity were observed. As shown in Figure 3.6a and b, although the levels of expression varied between cells, all D1 cells after 3 passages from initial labelling still expressed GFP, and maintained GFP expression at least for the next 20 passages.

Figure 3.5 CFDA SE- and QD-labelling of D1 MSCs. (a-d) D1 cells stained with CFDA SE. (a) Bright field and (b) fluorescent image of the D1 cells stained with CFDA SE directly after labelling. (c) Bright field and (d) fluorescent image of the D1 cells stained with CFDA SE after 5 days. (e-h) D1 cells stained with QDs. (e) Bright field and (f) fluorescent image of the D1 cells stained with QDs directly after labelling. (g) Bright field and (h) fluorescent image of

a 100µm c 100µm d 100µm 100µm b g 100µm h 100µm e 100µm f 100µm QD s la b e ll in g CF D A S E l a b e ll in g

Figure 3.6 GFP labelling of D1 cells using lentiviral transduction. (a) Bright field and (b) fluorescent image of the D1 cells transduced with GFP.

Although it has been demonstrated that GFP MSCs are not affected by the enforced expression of fluorescent proteins and maintain adipogenic and osteogenic potential as well surface expression marker profile (Ripoll and Bunnell 2009), there are reports describing adverse effects associated with GFP expression (Baens et al. 2006; Guo et al. 2007). For this reason, the differentiation potential of transduced D1 cells was verified. GFP D1 cells were induced to undergo adipogenesis and osteogenesis in the presence of the appropriate inductive media, as described earlier in section 3.2.1. It was found that the GFP D1 cells were able to undergo both adipogenic (Figure 3.7a-d) and osteogenic (Figure 3.8e-f) differentiation, as visualized by Oil Red and Alizarin Red staining, respectively, whereas no staining was detected in uninduced cultures.

Above, three different labelling methods for tracking of D1 cells have been described. As the assessment of the renogenic potential of MSCs will require formation of kidney chimeras using mouse kidney cells, D1 cells, which are of mouse origin, could not be detected with a species- specific antibody. Nevertheless, a species-specific antibody can enable discrimination between human MSCs and mouse cells. To test the suitability of a human anti-nuclear antibody, human MSCs were immunostained using the human anti-nuclei antibody and co-stained with DAPI

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(Figure 3.8a-c). No unspecific staining of human cells was detected when the primary antibody was omitted (Figure 3.8d-f). Further, the potential cross-reactivity of the antibody was tested on mouse cells. Using the protocol for formation of kidney chimeras described in the section 4.2.1 mouse embryonic kidneys were disaggregated and obtained kidney cells re-aggregated to form new kidneys in the absence of human MSCs. The re-aggregated mouse kidneys containing only mouse cells were stained subsequently with an antibody identifying expression of a nuclear kidney marker Wt1 and the antibody detecting human nuclei. Accordingly expression of Wt1 was observed; however, no expression of human nuclear antigen was found in the re-aggregated mouse kidney (Figure 3.9a-c). Some unspecific signal was detected in cytoplasm of the kidney cells.

In summary, several labelling methods were tested here for their suitability for imaging of MSCs, including labelling with CFDA SE, QDs, and GFP as well as staining with a species-specific antibody. For the purpose of this study labelling with QDs and viral transduction with GFP were identified as most suitable for tracking mouse MSCs. Furthermore the specificity of species- specific antibody for human MSC detection was assessed and subsequently proven to be suitable for human MSC tracking.

Figure 3.7 Confirmation of multilineage differentiation potential of GFP D1 cells (a) Stimulated with adipogenic inductive medium GFP D1 cells accumulate lipid vacuoles inside after 14 days of adipogenic induction. (b) GFP expression in induced towards adipocytes GFP D1 cells (in green). (c) Stimulated with adipogenic inductive medium GFP D1 cells display lipid vacuoles stained with Oil Red. (d) Oil Red staining of GFP D1 cells cultured in standard culture medium showed that no lipid vacuoles had formed. (e) Stimulated with osteogenic inductive medium GFP D1 cells show extracellular calcium deposits visualized using Alizarin Red staining after 14 days of osteogenic induction. (f) Alizarin Red staining of GFP D1 cells cultured in standard culture medium showed that no calcium deposits were present.

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Figure 3.8 Staining of human MSCs using an anti-human nuclei antibody. (a) Human MSCs labelled with anti- human antibody. (b) Labelled human MSCs co-stained with DAPI. (c) Merged image of a and b indicates that all cells are labelled. (d) Human MSCs incubated only with appropriate secondary antibody; the anti-human antibody was omitted. (e) Human MSCs co-stained with DAPI (in blue). (f) Merged image of d and e.

Figure 3.9 Assessment of specificity of anti-human nuclei antibody tested on mouse cells. (a) Staining using the antibody detecting human nuclei performed on the same re-aggregated kidney. (b) A re-aggregated mouse kidney stained with an antibody identifying expression of a nuclear kidney marker Wt1. (c) Merge.

b a 100µm 100µm 100µm Wt1 merge anti-human antibody c d e f 100µm 100µm 100µm 100µm 100µm 100µm c b a