Targeting small GTPase Cdc42 in aged adipose mesenchymal stem cells for the improvement of effectiveness of cell therapy for bone regeneration

Collaborative Research Grants Program 2020-2022

The purpose of the Project: Rejuvenation of aging adipose mesenchymal stem cells by Cdc42 inhibition for improving the therapeutic effectiveness of bone fracture repair
Accumulating evidences suggest that small Rho GTPase cell division control protein 42 homolog, also known as Cdc42 have a great impact on the pathogenesis of chronic and age-related diseases. Our previous studies have shown that a Cdc42 inhibitor CASIN increased proliferative activity and osteogenic differentiation potential in adipose-derived mesenchymal stem cells (ADMSCs) isolated from 24-month old rats.As a continuation of our previous work related to in vitro Cdc42 research, in the present project we are very interested in evaluating in vivo testing of bone healing induced by the activation of ADMSCs via Cdc42 regulation. We hypothesize that inhibition of aging-associated increase in Cdc42 activity in MSCs in ex-vivo conditions can improve MSC osteogenic differentiation and migration and reduce osteoclastic activity for MSC-based therapy in bone regeneration. We propose that a method based on the use of cell therapy for ADMSCs after ex-vivo inhibition of Cdc42 activity will be an effective approach for stimulating reparative osteogenesis and will improve the results of treatment of pathological bone fractures.

Main Results of the project for 2020:
For the first year of the project, all calendar tasks had been accomplished. MSCs were isolated and cultured from the rat adipose tissue (AD-MSCs). The mesenchymal nature of the isolated cells was confirmed through the surface markers’ expression profile – the presence of CD90 and CD105 and lack of CD34, CD45, and CD31. ADMSCs were able to differentiate down the adipogenic, chondrogenic, and osteogenic lineage and the differentiation was confirmed through the staining with Oil Red O, Alcian Blue and Alzarin Red correspondingly. The obtained cells were sorted using magnetic bead separation with CD105 antibody. Positively sorted cells after cultivation for 4–5 passages represented a fairly homogeneous population, in which about 90% of cells were positive for mesenchymal markers CD90 and CD105, and only a small part of cells expressed hematopoietic markers CD34, CD45 and CD31.
Another important task was aimed to evaluate the pharmacological inhibition and silencing of Cdc42 for markers of ADMSC proliferation and senescence. Pharmacological inhibition was performed with the small molecule CASIN. Stealth CDC42 siRNAs: RSS329925, RSS329926, RSS329927 (Ther-moFisherScientific) were used for silencing. The most effective was the use of the Stealth CDC42 siRNAs RSS329927 sequence. Both approaches improved proliferation and reduced the level of aging markers in ADMSCs isolated from the adipose tissue of old animals.
ADMSCs from old animals had upregulated levels of the cyclin-dependent kinase inhibitor p16INK4a and p21 compared to cells from young animals. Treatment with CASIN and siRNA reduced this expression to levels observed in cells obtained from animals 1 month of age.
CASIN and siRNA significantly reduced the percentage of SA-b-Gal-positive cells in ADMSC isolated from 24-month-old rats compared to controls. As an outcome of the productive year, 2 articles were submitted to peer-reviewed journals with impact factor.
Main Results of the project for 2021:
For the second year of the project, all calendar tasks had been accomplished. As a result of the studies, it was shown that the effect of the Cdc42 inhibitor with the small molecule CASIN and the silencing of Cdc42 with the help of a small interfering RNA leads to an increase in the indicators of osteogenic markers compared to the control. Thus, on the 14th day of osteogenic differentiation in the cells that was pretreated with CASIN and transfected with miRNA, the activity of alkaline phosphatase and collagen secretion increases, and on the 21st day, the accumulation of calcium increases in comparison with the untreated control. It has been shown that with aging ADMSC the secretion of osteoprotegerin decreases. The results obtained showed that exposure of the Cdc42 inhibitor to the small molecule CASIN and silencing of Cdc42 with the help siRNA leads to an increase in the secretion of osteoprotegerin. ADMSC conditioned medium from young cells decreased bone resorption activity of osteoclasts compared to old cells. The medium obtained from cells after exposure to the Cdc42 inhibitor with CASIN and silencing of Cdc42 had increased anti-osteoclastic activity compared to the untreated control. This indicates that inhibition of Cdc42 may enhance the anti-osteoclastic activity of MSCs and accelerate bone regeneration during cell transplantation.The effects of CASIN and silencing with siRNA on the old ADMSCs have been shown to enhance the migration process in the scratch assay compared to the untreated controls. Transwell migration assay showed that directional migration was higher in young cells, whereas in the group of old cells and CASIN-treated cells, the ability to migrate was reduced. At the same time, Cdc42 silencing with siRNA enhanced migration in old cells, which exceeded it even in young cells. Thus, the results obtained indicate that pharmacological inhibition and silencing of Cdc42 enhances stem cell migration, but only silencing enhances the migration directed to the chemoattractant. Thus, we can conclude that the inhibition of Cdc42 can improve osteogenic differentiation, anti-osteoclastic activity and migration of aged mesenchymal stem cells from adipose tissue. Research results were presented at an international conference Cell Bio Virtual 2021, 1-10 December 2021.
Main Results of the project for 2022:
To evaluate the regenerative potential of mesenchymal stem cells modified with the small molecule CASIN and siRNA silencing, a model of induced ulna fracture in rats with ulnar shaft osteotomy was created. During the indicated period, a surgically induced fracture of the ulna was modelled in 40 laboratory animals for 4 groups of animals, including the control group and 3 experimental ones (old MSCs, old MSCs + CASIN, old MSCs + siRNA). 
Analysis of relative bone mineral density at 4, 8, 16 and 24 weeks revealed consistent increase in bone regeneration process in group of animals that received MSCs transfected with siRNA up to 90% after 16 weeks and 58% at final 24 weeks and MSCs+CASIN up to 44% at 16 weeks compared to the negative dynamics of bone density in Control group of animals. Survival of cells was confirmed with LUC-LVT transfection and monitored in luminescent field of In Vivo Imaging System Spectrum CT (Caliper, USA). The results were also confirmed through the histological analysis and stained with H&E. In groups with siRNA and CASIN prominent osteogenesis was monitored and active substitution of formed fibrous callus with newly formed bone tissue.
As a result of performed studies it was shown that the approach based on the inhibition of Cdc42 activity in aged mesenchymal stem cells improves their therapeutic potential in bone regeneration in the area of fracture healing in vivo.
Research results were presented at an international conference: Herbert Fleisch Workshop in Brugge, Belgium, 20-22 November 2022 and an international conference «ГОРИЗОНТЫ СОВРЕМЕННОЙ ТРАВМАТОЛОГИИ И ОРТОПЕДИИ» (“HORIZONS OF MODERN TRAUMATOLOGY AND ORTHOPEDICS”), Turkestan, 15-16 September 2022