Sclerostin-silenced human umbilical cord mesenchymal stem cells ameliorate bone metabolism in steroid-induced femoral head necrosis

Figure 3 Phenotypic identification and multipotent differentiation of human umbilical cord mesenchymal stem cells and human umbilical cord mesenchymal stem cells with sclerostin-silenced. Characterization of human umbilical cord mesenchymal stem cells and human umbilical cord mesenchymal stem cells with sclerostin-silenced. A and B: Flow cytometry analysis of the two cell groups demonstrated typical expression of mesenchymal stem cell surface markers: CD73, CD90 and CD105 were highly expressed (≥ 95%), while hematopoietic markers CD34, CD11b and CD45 showed minimal expression (≤ 5%), confirming mesenchymal stem cell phenotype; C-E: Multilineage differentiation potential of human umbilical cord mesenchymal stem cells (hUCMSCs); F-H: Multilineage differentiation potential of hUCMSCs with sclerostin-silenced (sh-hUCMSCs); I: Alizarin Red staining of both cell groups demonstrated upregulation of osteogenic markers (Runt-related transcription factor 2, alkaline phosphatase, and osteoprotegerin), confirming osteogenic differentiation, with sh-hUCMSCs exhibiting a stronger osteogenic potential; J: Oil Red O staining of both cell groups demonstrated upregulation of adipogenic markers (peroxisome proliferator-activated receptor gamma, adipocyte protein 2, and CCAAT/enhancer-binding proteins), confirming adipogenic differentiation. Notably, sh-hUCMSCs exhibited a reduced adipogenic potential compared to the hUCMSCs group; K: Alcian Blue staining of both cell groups revealed upregulation of chondrogenic markers (SRY-box transcription factor 9, type II collagen alpha 1 chain, and aggrecan), confirming chondrogenic differentiation. Notably, sh-hUCMSCs exhibited enhanced chondrogenic potential compared to the hUCMSCs group. All mRNA expression data represent three independent experiments (mean ± SEM). ^aP < 0.05 vs human umbilical cord mesenchymal stem cells; ^bP < 0.01 vs human umbilical cord mesenchymal stem cells. hUCMSCs: Human umbilical cord mesenchymal stem cells; sh-hUCMSCs: Human umbilical cord mesenchymal stem cells with sclerostin-silenced; ALP: Alkaline phosphatase; OPG: Osteoprotegerin; RUNX2: Runt-related transcription factor 2; PPARγ: Peroxisome proliferator-activated receptor gamma; C/EBPs: CCAAT/enhancer-binding proteins; AP2: Adipocyte protein 2; SOX9: SRY-box transcription factor 9; COL2A1: Type II collagen alpha 1 chain; ACAN: Aggrecan.