Optimization of lentiviral transduction parameters and its application for CRISPR-based secretome modification of...

Mesenchymal stem cells (MSCs) hold a great promise for successful development of regenerative medicine. Among the plenty of uncovered MSCs sources, desquamated endometrium collected from the menstrual blood probably remains the most accessible. Though numerous studies have been published on human endometrium-derived mesenchymal stem cells (hMESCs) properties in the past years, there are only a few data regarding their genetic modulation. Moreover, there is a lack of information about the fate of the transduced hMESCs. The present study aimed to optimize hMESCs transduction parameters and apply Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology for genome and secretome modification. The fate of hMESCs transduced either in presence of polybrene (Pb) or protamine sulfate (Ps) was assessed by alterations in CD expression profile, growth rate, cell size, migration capability, osteogenic, adipogenic, and decidual differential potentials. Here, we postulated that the use of Ps for hMESCs genetic manipulations is preferable, as it has no impact on the stem-cell properties, whereas Pb application is undesirable, as it induces cellular senescence. Plasminogen activator inhibitor-1 was selected for further targeted hMESCs genome and secretome modification using CRISPR/Cas9 systems. The obtained data provide optimized transduction scheme for hMESCs and verification of its effectiveness by successful hMESCs genome editing via CRISPR/Cas9 technology.