Isolation of Human BAMBI<sup>high</sup>MFGE8<sup>high</sup> Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBI^highMFGE8^high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBI^highMFGE8^high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBI^highMFGE8^high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBI^highMFGE8^high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBI^highMFGE8^high UC-MSCs.