DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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Figure 7 - A) Immunofluorescence images of Beta-III tubulin and NFH in branched extensions of MSCs on
soft matrices (E ≈ 1 kPa). Scale bars are 5 mm. B) B-III tubulin, NFH, and P-NFH all localize to the branches
of MSCs on the softest substrates with E < 1 kPa (mean ± SEM). Nestin, B-III tubulin, MAP2, and NFL
Western blotting (inset) confirms expression only on soft gels (GL = Glass). Adapted from Engler et al.,
2006.
Intensity analyses of immunofluorescence images as well as Western blots (Figure 7)
confirm that proteins markers for neural commitment are expressed only in cells on
the softest matrices. Protein markers for neuronal commitment (nestin), immature
neurons (B-III tubulin), mid/late neurons (microtubule associated protein 2; MAP2),
and even mature neurons (NFL, NFH, and P-NFH) (Engler et al., 2006). These results
from Engler et al. 2006 suggest that this process that occurs with MSCs somehow
recapitulates what is seen during neurogenesis in vivo, as seen in more complex
microenvironments like the brain (Kondo et al., 2005; Wislet-Gendebien et al., 2005).
Furthermore, when looking across the range of matrix stiffness, to the
cytoskeletal markers and transcription factors characteristic of the distinct lineages
(Figure 8) it is indicated that MSCs lineage specification occurs (process similar to an
incomplete differentiation), being consistent with the lineage profiling of Figure 5 and
6. On the softest, neurogenic matrices, a majority of cells express B-III tubulin, which,
along with P-NFH and NFH, is visible in long, branched extensions but is poorly
expressed, if at all, in cells on stiffer gels (Figure 7). On moderately stiff, myogenic
matrices, MSCs upregulate the transcription factor MyoD1, localizing it to the nucleus
(Figure 8) (Engler et al., 2006).