Mesenchymal Stromal Cells for Sphincter Regeneration: Role of Laminin Isoforms upon Myogenic Differentiation

<div><p>Multipotent mesenchymal stromal cells (MSCs) are well known for their tri-lineage potential and ability to differentiate <i>in vitro</i> into osteogenic, chondrogenic or adipogenic lineages. By selecting appropriate conditions MSCs can also be differentiated <i>in vitro</i> into the myogenic lineage and are therefore a promising option for cell-based regeneration of muscle tissue such as an aged or damaged sphincter muscle. For the differentiation into the myogenic lineage there is still a need to evaluate the effects of extracellular matrix proteins such as laminins (LM) which are crucial for different stem cell types and for normal muscle function. The laminin family consists of 16 functionally different isoforms with LM-211 being the most abundant isoform of adult muscle tissues. In the sphincter tissue a strong expression of the isoforms LM-211/221, LM-411/421 and LM-511/521 can be detected in the different cell layers. Bone marrow-derived MSCs in culture, however, mainly express the isoforms LM-411 and LM-511, but not LM-211. Even after myogenic differentiation, LM-211 can hardly be detected. All laminin isoforms tested (LM-211, LM-411, LM-511 and LM-521) showed a significant inhibition of the proliferation of undifferentiated MSCs but, with the exception of LM-521, they had no influence on the proliferation of MSCs cultivated in myogenic medium. The strongest cellular adhesion of MSCs was to LM-511 and LM-521, whereas LM-211 was only a weakly-adhesive substrate for MSCs. Myogenic differentiation of MSCs even reduced the interaction with LM-211, but it did not affect the interaction with LM-511 and LM-521. Since during normal myogenesis the latter two isoforms are the major laminins surrounding developing myogenic progenitors, α5 chain-containing laminins are recommended for further improvements of myogenic differentiation protocols of MSCs into smooth muscle cells.</p></div

Expression of laminin chains in the mini-pig sphincter tissue.

<p>Cryostat sections of an isolated mini-pig sphincter tissue were either stained with hematoxylin-eosin (HE) or labeled with laminin chain specific antibodies (red) together with an antiserum against von Willebrand factor (vWF; green) labeling endothelial cells. By HE staining the inner epithelial lining (E), mucosa, longitudinal smooth muscle layer (LM) and circular smooth muscle layer (CM) can be distinguished. The laminin α chains (α2, α4, α5), β chains (β1, β2) and the γ1 chain are expressed in the mucosa, in the different muscular layers and around endothelial cells, albeit with different intensities. In the merged pictures the weaker expression of the laminin α2, β1 and γ1 chains in some endothelial cells is hidden behind the strong signal for von Willebrand factor staining. Cell nuclei were counterstained in blue with DAPI (bar: 500 μm).</p

Influence of recombinant laminin isoforms on the proliferation rate of MSCs.

<p>MSCs in early passages were grown for seven days in expansion medium (GMP+<b>)</b> with or without 10 μg/ml of the recombinant laminin isoforms LM-211, LM-411, LM-511 and LM-521. All laminin isoforms decreased the proliferation rate of MSCs. In contrast, cultivation of MSCs in myogenic differentiation medium with the different recombinant laminin isoforms had no significant effect on the proliferation rate with LM-521 being the exception (n = 3 donors; experiments performed in duplicates; error bars indicate standard error of the mean; one-way ANOVA analysis; **p<0.01; ***p<0.001 in comparison to control).</p

Laminin expression by primary MSCs, the cell line HITB5 and tissue-derived HBdSMC.

<p>RT-PCR analyses (A) and immunofluorescence staining (B) of undifferentiated MSCs (Undiff), myogenically differentiated MSCs (Myo), the cell line HITB5 and HBdSMC in early passages suggested the expression of several laminin isoforms. The highest expression was observed for the α4, α5, β1 and γ1 chains. The α2 chain was only weakly expressed. Cell nuclei were counterstained in blue with DAPI (bars: 50 μm).</p

Cell adhesion to plastic-immobilized laminin isoforms.

<p>Cell-matrix interactions with undifferentiated MSCs (Undiff), myogenically differentiated MSCs (Myo), the cell line HITB5 and HBdSMC were quantitatively determined by single cell force measurement (n = 9 cells) and qualitatively by spotting assays on different recombinant laminin isoforms. LM-511 and LM-521 were the strongest adhesive substrates for smooth muscle cells and undifferentiated MSCs. Myogenic differentiation diminished the binding of MSCs to LM-411. LM-211 was only a weakly-adhesive substrate for smooth muscle cells and MSCs, and upon myogenic differentiation the adhesive capacity of LM-211 was further diminished (bar: 200 μm; error bars indicate standard error of the mean; one-way ANOVA analysis; *p<0.05; **p<0.01; ***p<0.001 in comparison to HITB5).</p