2. DESARROLLO
2.6. DIAGNÓSTICO DE LOS TRASTORNOS DE LA LECTURA
that gene, does not necessarily correspond to it having a function, especially when that expression is very low (Chelly et al., 1989). POU5F1, NANOG and TERT expression in hES cells are known to have a physiological outcome, so comparing expression of these genes in hES to that in other cell types would give an indication as to whether the expression level in the other cell types is functionally relevant.
To estimate the significance of previous reports of POU5F1, NANOG and TERT expression in fMSC and fMSC-like cells, the expression of these genes was measured in a selection of fMSC and hES lines (see Table 5.2 for full description of
cell lines) using quantitative RT-PCR. The analysis of these undifferentiated, subconfluent fMSC and undifferentiated hES cells was carried out in parallel with differentiated derivatives of both cell types: fMSC differentiated to bone (‘fMSC BONE’), and hES cells differentiated to embryoid bodies, (‘hEB’). Should expression of any of the factors be functional in fMSC in the same manner as in hES cells, further differentiation would then be expected to correspond with a reduction in expression levels. Additionally, fMSC grown to confluence (‘fMSC CONF’) have less growth and differentiation potential than counterparts maintained in a subconfluent state, and so would also be expected to express less POU5F1, NANOG and TERT than subconfluent cells (Guillot et al., 2006a). Primary fibroblasts were used as a negative control, and an adult peripheral blood leukocyte (‘PBL’) sample was also analysed. Both panels (i) and (ii) of each of Figure 4.1 to Figure 4.3 show the 2^-dCT plotted against the cell type, where 2^-dCT is directly proportional to the amount of transcript present, based upon the difference (d) in qPCR cycle number (CT) required to meet a threshold (0.2) between the gene of interest and GAPDH.
(i)
(ii)
Figure 4.1. POU5F1 expression in fMSC and hES cells
Panel (i) shows the expression of POU5F1 in two hES cell lines, hEBs, three fMSC lines, plus one CONF sample and one sample differentiated to bone. Mean hES cell expression is 18 fold higher (0.082/0.005) than that of hEBs, and 410 fold higher (0.082/0.0002) than the most highly expressing fMSC line (fBM12+4). Panel (ii) excludes hES cells from the graph to show more clearly the expression patterns in other cells. POU5F1 expression in fMSC is highly variable, not significantly
decreased in either fMSC grown to confluence of differentiated to bone, and actually less than in primary fibroblasts, a terminally differentiated cell type.
(i)
(ii)
Figure 4.2 NANOG expression in fMSC and hES cells
Panel (i) shows the expression of NANOG in two hES cell lines, hEBs, three fMSC lines, plus one CONF sample and one sample differentiated to bone. Mean hES cell expression is five fold higher (0.043/0.0083) than that of hEBs and 81 fold higher (0.043/0.00053) than the most highly expressing fMSC line (AF MSC). Panel (ii) excludes hES cells from the graph to show more clearly the expression patterns in other cells. NANOG expression in fMSC is not as variable as POU5F1 expression, where although AFMSC is almost five fold higher than the other fMSC lines, the other three measured are very similar. The difference in expression of NANOG between hES and these three lines is similar to that of POU5F1, at 390 fold (0.043/0.00011). Expression in these three lower expressing lines, fBL12+4, fLiv10+4 and fBl8+3 is the same as that found in fibroblasts and in fMSC grown to confluence, and much lower (five fold) than that found in cells differentiated to bone and in adult PBL.
(i)
(ii)
Figure 4.3 TERT expression in fMSC and hES cells
Panel (i) shows the expression of TERT in two hES cell lines, hEBs, three fMSC lines, plus one CONF sample and one sample differentiated to bone. Mean hES cell expression is 6.6 fold higher (0.0019/0.00028) than that of hEBs and 45 fold higher (0.0019/0.000042) than the most highly expressing fMSC line (fBM12+4). Panel (ii) excludes hES cells from the graph to show more clearly the expression patterns in other cells. TERT expression in fMSC is again very variable between fMSC, as for POU5F1, and no pattern exists to distinguish fMSC lines from either their differentiated derivatives or primary fibroblasts.
From these results it can be concluded that the expression of pluripotency markers POU5F1, NANOG and TERT in fMSC is firstly very low and secondly non-functional, at least for the functions considered canonical for these transcripts (Thomson et al., 1998; Chambers et al., 2003; Loh et al., 2006). There are vast
differences between the expression of each of these genes between undifferentiated hES cells and undifferentiated fMSC. The expression of each marker is shown to directly correspond with, and contribute to, the novel pluripotentiality and division potential of hES cells, and so expression at a level so vastly decreased from this is not indicative of parallel transcript functionality in the other cell types (Brehm et al., 1997; Chambers et al., 2003; Niwa et al., 2005). Additionally, the magnitude of these expression differences is greater than that between hES and hES differentiated to hEBs, the latter of which are characterised by their down-regulation of these markers.
Differentiation of fMSC, or their growth to confluency, does not cause a reduction in transcript levels of any of the genes, and in the cases of TERT and NANOG, the level of transcript was increased in the cells differentiated to bone compared to every fMSC sample. The variability of expression levels of TERT, NANOG and POU5F1 between the different fMSC cell lines, and the absence of any trend linking their expression to cells with different differentiation or growth potential (i.e. fMSC compared to fibroblasts) is further indication that the actual level of transcript is so low that it can be considered spurious. The lack of expression of so-called pluripotency markers does not detract from the importance and value of fMSC for the treatment of mesodermal diseases such as osteogenesis imperfecta and muscular dystrophy, or of injury trauma (Chan et al., 2006; Guillot et al., 2008).
The transcriptional network active in hES cells to promote a pluripotent, immortal phenotype is not active in fMSC. If fMSC have the capacity to differentiate between germ layers, this is facilitated by a separate transcriptional pathway. The differentiation capacity of fMSC should be characterised by appropriate experiments, using clonal dilution of the cells. This is an important experiment, as they are a useful and easily grown source of cells.
C C h h a a p p t t e e r r 5 5 Comparison of genomic imprinting in fMSC
and hES cells
5.1