Bone Marrow Mesenchymal Stem Cells Adhesion Assay

Yang Z. and Xiao R.
Bio-protocol, 2016

Here, the authors present a protocol for culture of bone marrow MSC (BM-MSCs) on laminin-521 or laminin-511. The protocol is based on the method by Siler et al., 2000, and can easily be translated to MSCs from other origin or alternative ECMs coating. Both laminin isoforms show a significantly better efficient attachment compared to uncoated wells and also support seeding of a lower cell number compared to uncoated plats. The BM-MSCs adhere to the laminin-coated wells within 10 min, while for non-coated wells, it may take longer time. In this protocol, a final coating concentration of 2 μg/cm2 is used but can effectively be lowered 4-10 times without loss of function.

 

Laminin-521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light-induced Cell Sheet Technology

Jiang Z., Xi Y., Lai K., Wang Y., Wang H., Yang G.
BioMed Research International, 2016

The cell sheet technology is an area of research that is of great interest for tissue engineering and regenerative medicine. Here, the authors investigated the effects of an ECM coating on rat bone marrow mesenchymal stem cells (rBMSC) cultured on light-induced TiO2 nanodot films. Cell sheets can be detached on a TiO2 nanodot-coated quartz substrate by using UV365 illumination. The effects of rat fibronectin, human recombinant laminin-521, -511, -421, and -111 on the formation of cell sheets were investigated and also compared to uncoated films. The result showed the highest success for laminin-521. rBMSCs rapidly attached and spread on films coated with laminin-521 (1.2 ug/ml) and formed intact cell sheets after 5 days of culture. Laminin-521 promote the formation of rBMSC sheets with good viability under hyperconfluent conditions (4 to 8 layers of cells). The cells also maintained multilineage potential, including osteogenic, adipogenic, and chondrogenic differentiation. The cell sheets formed had rich ECM (including collagen I) and cells were connected with each other in a dense network-like tissue. rBMSC cultured on uncoated surface and cells partially detached and failed to form cell sheets. In summary, laminin-521 and UV365 illumination systems provided a simple, rapid, and effective cell sheets strategy.

 

CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion and Migration of Human Mesenchymal Stem Cells

Yang Z., Dong P., Fu X., Li Q., Ma S., Wu D., Kang N., Liu X., Yan L., Xiao R.
Stem Cells, 2015

Here, we studied the role of CD49f (also known as integrin α6) in bone marrow MSCs. CD49f is preferentially expressed in fetal cells rather than adult cells, CD49f‐positive BM-MSCs possess higher CFU‐F formation ability and differentiation potential than CD49f negative cells, and the CD49f expression of BM-MSCs gradually decreases during in vitro passaging. An adhesion assay showed strong adhesion of BM-MSCs to both laminin 511 and 521 that were significantly higher than the control group coated with BSA, and the adhesion occurred evenly throughout the well. Pre‐blocking of CD49f on BM-MSCs inhibited the adhesion of fetal BM-MSCs to laminin 511 and 521. Also, CD49f knockdown dramatically decreased the differentiation of BMSCs. Inflammation (TNF-a) down-regulated CD49f in BMSCs with impaired differentiation, decreased adhesion to laminins and increased migration. This study provide evidence for CD49f as a stemness marker of BMSCs which is correlated with cell adhesion on laminin-521 and -511.

 

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

Seeger T., Hart M., Patarroyo M., Rolauffs B., Aicher W.K., Klein G.
PLOS ONE, 2015

In the sphincter tissue (smooth muscle) 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.