A Small-Molecule Cocktails-Based Strategy in Culture of Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells (MSCs) have a variety of unique properties, such as stem cell multipotency and immune regulation, making them attractive for use in cell therapy. Before infusion therapy, MSCs are required to undergo tissue separation, purification, and expansion in vitro for a certain duration. During the process of in vitro expansion of MSCs, the influence of culture time and environment can lead to cell senescence, increased heterogeneity, and function attenuation, which limits their clinical applications. We used a cocktail of three small-molecule compounds, ACY (A-83–01, CHIR99021, and Y-27632), to increase the proliferation activity of MSCs in vitro and reduce cell senescence. ACY inhibited the increase in heterogeneity of MSCs and conserved their differentiation potential. Additionally, ACY maintained the phenotype of MSCs and upregulated the expression of immunomodulatory factors. These results suggest that ACY can effectively improve the quantity and quality of MSCs.

Improving the immunomodulatory function of mesenchymal stem cells by defined chemical approach

“…Many studies reported the optimal combination of cytokines could improve the therapeutic effect of MSC, such as TNF-α and IFN-γ ( 44 ), IL-10 ( 45 ), HGF ( 46 ), or IDO ( 47 ). Our previous study also found that a cocktail of small-molecule compounds SM-ACY could improve the growth of MSCs ( 23 ). Moreover, we further discovered that SM-ACY which involving in three different signaling pathways respectively were capable to enhance the immunoregulatory effects of MSCs in vitro/vivo .…”

“…Following the Clinical Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University and written informed patient consent for tissue collection, MSCs were isolated following the protocol described in the previous study ( 23 ). In brief, fat tissues were isolated via liposuction from healthy patients defined as no history of malignancy or autoimmune deficiency.…”

“…Specific combinations of small molecule compounds were reported contribute to the maintenance of the pluripotency of stem cells and allow for the stable culturing of various types of stem cells ( 20 – 22 ). One of our previous studies indicated that stimulation of MSCs for a certain period of time with the compound cocktail ACY could effectively promote the viability of MSCs and maintain their differentiation potential ( 23 ), which demonstrated that ACY is a potential MSC modification material. In this study, we conducted a comprehensive analysis of immunomodulatory biological functions of MSCs against ACY.…”

Development of an in vitro human alveolar epithelial air-liquid interface model using a small molecule inhibitor cocktail

“…Y-27632 is a ROCK inhibitor [ 21 ], A-83-01 is an inhibitor of transforming growth factor-β (TGF-β) signaling [ 22 ], and CHIR99021 is an activator of Wnt signaling via inhibition of glycogen synthase kinase-3 [ 23 ]. YAC has been utilized in various combinations to culture human stem cells [ 24 ], and previous studies used at least one of these inhibitors to maintain proliferation of primary human AECIIs [ 11 , 12 , 25 ]. We obtained primary human AECs and initially cultured them in flasks containing medium supplemented with YAC under submerged conditions, followed by culturing at the air-liquid interface (ALI).…”