Citation

  • Authors: Passanha FR. et al.
  • Year: 2022
  • Journal: Stem Cells
  • Applications: in vitro / DNA, shRNA plasmid / PEIpro
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

shRNA lentiviral transduction - The plasmid pLKO.1 containing short hairpin RNA (shRNA) sequences targeting cadherin11 was obtained from Sigma-Aldrich together with a scrambled negative control. These plasmids were co-transfected with third generation lentiviral packaging and envelope vectors, pMD2.G, pRSV-Rev and pMDLg/pRRE (Addgene plasmids #12259, #12253 and #12251, respectively, which were gifts from Didier Trono31), into HEK-293T cells using PEIpro transfection reagent. - Lentiviral particles were harvested 48 and 72 h after transfection. - Five milliliters of viral supernatant were used to transduce hMSCs seeded at 5000 cells/cm2 in a T225 flask and incubated for 48 h. Transduced cells were selected with 2 µg/ml puromycin dihydrochloride (Sigma‐ Aldrich) in growth medium for 7 days and were then used for subsequent experiments.

Abstract

Controlling stem cell fate is the cornerstone of regenerative medicine. Cadherins have an important role in cell fate commitment and the function of cadherin-11 in the regulation of differentiation in human mesenchymal stem cells (hMSCs) has recently come to light. To better understand how cadherin-11 regulates hMSC behavior, we explored its interaction with receptor tyrosine kinases (RTK), an important family of proteins involved in a myriad of cellular functions. In this study, we provide evidence that cadherin-11, a cell adhesion protein expressed in hMSCs, regulates the activity of several RTKs, including PDGFRβ and PDGFRα. By knocking down cadherin-11 we found that the changes in the RTK activity caused hyperactivation of the MAPK pathways, which were sustained through the phosphorylation and nuclear translocation of ERK1/2 and subsequently caused a decrease in cell proliferation. Together these results provide compelling evidence for the important role of the interaction of cadherin-11 and RTKs in the behavior of hMSCs.

 

Pubmed