Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells

Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1⁺ LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1⁺ LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato⁺ LBM-like cells are defined as CD44^high CD140B^high CD49f⁻. Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44^high CD140B^high CD49f⁻ PRRX1⁺ LBM-like cells.