The most reliable markers for identifying mesenchymal stem cells (MSCs) in vitro, as established by the International Society for Cellular Therapy, rely on a combination of specific cell surface antigens, plastic adherence, and multipotent differentiation potential, ensuring accurate characterization across sources like bone marrow, adipose tissue, or umbilical cord. MSCs must express CD73 (ecto-5'-nucleotidase), CD90 (Thy-1), and CD105 (endoglin) at high levels (≥95% positive) while showing minimal expression (≤2% positive) of hematopoietic and endothelial markers such as CD34, CD45, CD11b, CD14, CD19, CD79α, and HLA-DR, detected via flow cytometry with fluorescently labeled antibodies for specificity. Additional markers like CD29 (integrin β1), CD44 (hyaluronic acid receptor), and CD166 (ALCAM) are often expressed, enhancing identification, though they are less specific. MSCs must adhere to plastic under standard culture conditions and demonstrate differentiation into osteoblasts, adipocytes, and chondroblasts, confirmed by assays like Alizarin Red, Oil Red O, and Alcian Blue staining, respectively. Gene expression analysis using RT-PCR or RNA sequencing can detect transcripts like RUNX2, PPARG, or SOX9 to validate differentiation potential, though these are secondary to surface markers for basal identification. Functional assays, such as colony-forming unit-fibroblast tests, assess proliferative capacity, while immunomodulatory properties like T-cell suppression support characterization but are less standardized. Variability in marker expression due to tissue source, passage number, or culture conditions necessitates combining multiple markers and functional tests, using standardized protocols and validated antibodies to ensure reproducibility, with some sources like adipose-derived MSCs showing higher CD36 or bone marrow MSCs expressing STRO-1 in early passages.

The easier and universal marker is Vimentin.

Depending on what kind of mesenchymal cells do you have and what methods you use, maybe you need some other markers.

Answer from Abdulhamid looks like typical ChatGPT answer.