Formulación por Componentes
BARRIO CALLES CARRERAS NORTE SUR ESTE OESTE
4. INSTITUCIONES Y ENTIDADES PUBLICAS PRESENTES EN EL MUNICIPIO
A number of labelling methods for MSCs was described in this chapter. Subsequently, D1 cells were labelled with CFDA SE, QDs and GFP, whereas human MSCs were stained with human specific antibody in order to elucidate the best labelling method for MSCs. All of the techniques have been employed previously in imaging of MSCs, as described in the introduction for this chapter.
According to results presented here, QDs have proven to be a more persistent labelling agent than CFDA SE, since CFDA SE could not be detected in D1 cells with fluorescent microscopy after 5 days of culture. CFDA SE loss of intensity, which led to exclusion of this labelling method from further experiments, has been demonstrated before in sheep MSCs after 8 days of culture (Weir et al. 2008). The loss of CFDA SE signal in D1 cells most likely occurred due to the high proliferation rate of the cells, as it was described that the intensity of this dye is reduced by half with every cell division (Lyons and Parish 1994).
In contrast, cells labelled with QDs remained stained after 5 days of culture. The localisation of QDs within D1 cells was perinuclear. QDs were shown previously to localise around the nucleus
found in the perinuclear region represent aggregated nanoparticles in endosomal vesicles (Muller- Borer et al. 2007). Importantly, no loss of intensity of the QD-labelling occurred during the culture period. Nevertheless, fewer nanoparticles were detected inside the D1 cells after 5 days of in vitro culture than when observed directly after labelling. This is in agreement with Rosen et al, who showed progressive loss of QDs in human MSCs (Rosen et al. 2007). Loss of QD labelling was also reported in mouse embryonic stem cells (ESCs) (Lin et al. 2007; Pi et al. 2010) and mouse embryonic fibroblasts (MEFs) (Pi et al. 2010). In particular ESCs were demonstrated to rapidly lose the labelling within a few days of in vitro culture (Lin et al. 2007). MEFs are more likely to retain QD-labelling, since the decrease in the number of labelled cells over a period of time was smaller in MEFs than in ESCs (Pi et al. 2010). It was suggested that loss in QD- labelling in ESCs is not due to cell division, as treatment with mitomycin C which inhibited the proliferation of the cells did not prevent loss of labelling. Accordingly other mechanisms such as degradation and excretion of QDs were proposed. At the same time inhibition of proliferation did help to retain the labelling in MEFs; implying that proliferation rate of the cells may be linked to loss of QD-labelling and that there exist different mechanisms for QDs loss in different cells (Pi et al. 2010). In contrast, human MSCs were shown to retain the QDs for up to 44 days in culture (Rosen et al. 2007). As QD-labelled human MSCs were observed to divide only a few times during this period of time (Rosen et al. 2007), the retention of QDs in human MSCs might be also attributed to lower proliferation rate of the cells. D1 cells used in this study seemed to lose QDs more rapidly than described by Rosen et al. human MSCs. Although no direct comparison between QD-labelled D1 cells and human MSCs was attempted, it might be possible that D1 cells lose QDs more quickly due to a higher proliferation rate. D1 cells used in this study were robustly proliferating and therefore passaged regularly every 2-3 days in comparison to primary
human MSCs used also in this study which displayed a much lower proliferation rate and accordingly were sub-cultured approximately once a week.
An important issue regarding QDs is the potential transfer of nanoparticles from QD-loaded MSCs to other cells. It has been demonstrated that QDs can be transferred to other cells. Supernatants collected from the cultures of ESCs labelled with QDs were shown to contain QDs. Subsequently QDs derived from such supernatants were used to label MEFs in the presence of a labelling buffer (Pi et al. 2010). Nevertheless, Rosen et al. demonstrated that human MSCs labelled with QDs did not transfer QDs to unlabelled MSCs. Furthermore, no uptake of QDs by cardiac myocytes from mechanically disrupted QD-labelled human MSCs occurred (Rosen et al. 2007). Another study on QD-labelled rat MSCs showed that QDs are not transferred when co- cultured with cardiac myocytes (Muller-Borer et al. 2007). The transfer of QDs between the cells has not been addressed in this study.
There are a number of reports describing the use of GFP-labelled MSCs (Min et al. 2002; Lu et al. 2005; Fukui et al. 2009). Transduction with GFP have been shown to be an ideal method for long term tracking of bone marrow-derived stem cells, namely hematopoietic stem cells (HSCs) (Tao et al. 2007) and MSCs (Lu et al. 2005). In the current study the D1 cells were transduced with lentiviral particles carrying enhanced GFP to induce constitutive GFP expression in MSCs. GFP D1 cells maintained labelling over a long period of time following many passages. However, GFP expression might have some adverse effects on the cells (Baens et al. 2006; Guo et al. 2007). It has been shown that expression of enhanced GFP or GFP fusion proteins inhibits NF-κB and JNK signalling pathways in a human embryonic kidney cell line (Baens et al. 2006). Furthermore GFP transgenic mouse expressing the fluorescent protein under the control of β-
actin promoter were shown to display renal defects including increase in glomerular extracellular matrix, occasional mesangiolysis, and tubulointerstitial injury, which are accompanied by proteinuria. As it has been confirmed that the insertion of the transgene encoding GFP did not disrupt nor modify expression of adjacent genes, the authors suggested that high expression levels of GFP in the glomeruli might be responsible for the observed defects (Guo et al. 2007). In order to examine if stable GFP expression does not negatively affect D1 cells and hence alter their differentiation potential, GFP D1 cells were confirmed to undergo adipogenic and osteogenic degeneration in standard differentiation assays. The obtained results were in agreement with previous report showing that that expression of GFP in MSCs does not alter their adipogenic and osteogenic potential (Ripoll and Bunnell 2009). Nevertheless, it is difficult to speculate if GFP expression is going to influence D1 cells behaviour in the chimeric kidneys thus if GFP D1 cells will have similar differentiation potential towards kidney-specific phenotype as non-transduced cells.
Another concern regarding GFP labelling of D1 cells is that the GFP expression might be downregulated when the labelled cells start to differentiae into kidney-like cells making the detection of D1 cells impossible. None of the GFP transgenic mice was shown to ubiquitously express GFP in all its tissues. Accordingly transgenic mice expressing GFP under the control of a human ubiquitin C promoter showed absence of GFP signal in the renal tissue (Swenson et al. 2007). There is also a discrepancy in levels of GFP expression between different GFP transgenic mice in the same tissues. Different percentage of peripheral blood cells was detected to be positive for GFP in different transgenic mice (Swenson et al. 2007). It is therefore not clear if GFP D1 cells will not loss the expression when undergoing differentiation. In this study it was
demonstrated that GFP expression is maintained upon adipogenic differentiation of D1 cells, which implicates that GFP D1 cell, might continue the express GFP when differentiating into renal structures.
Finally, horizontal transfer of labels between cells via microvesicles should be taken in consideration. Microvesicles mediate intracellular communication by delivering proteins and mRNA between cells. Recently, it was demonstrated that microvesicles derived from human MSCs protect mice from tubular damage in glycerol-induced acute kidney injury (Bruno et al. 2009). However, at the same time it has been shown that cultured tubular cells can take up microvesicles labelled with PKH-26 dye and subsequently become labelled with PKH-26 (Bruno et al. 2009). Therefore, there also exists the possibility that GFP mRNA can be transferred to kidney cells resulting in inaccurate GFP labelling of other cells than MSCs.
In particular, for detection of human primary MSCs a staining for human antigen can be performed. It was demonstrated that human specific antibodies may be used for detection of human primary MSCs in fixed samples obtained from rats (Azizi et al. 1998; Jeong et al. 2009). Here the staining with human anti-nuclei antibody has been demonstrated as a suitable method for labelling human cells.
In conclusion, QDs have proven to be better labelling agent than CFDA SE, although some loss of QD-labelling did occur during the in vitro culture period. Nevertheless, D1 cells labelled with QDs retained sufficient nanoparticles to be used for imaging, allowing sensitive detection over a period of 5 days. Therefore QDs can be used to investigate the renogenic potential of D1 cells in short-time experiments. Genetic modification using lentiviral transduction of D1 cells provided a
cells. However, since it was described that GFP expression in the kidneys of transgenic mice led to renal defect it is unknown if GFP expression in this setting might not also influence the course of MSC differentiation. For this reason two labelling methods, QDs and GFP, were chosen for tracking D1 cells in the chimeric kidneys. For human MSCs antibody staining against human nuclear antigen will be used to track the cells.