[Background] To achieve the disease modeling of skeletal dysplasia, human induced pluripotent stem cells (iPSCs) will be useful but the induction method for limb bud mesenchymal (LBM) cells, which can give rise to most of the future limb elements - bone, cartilage and tendon/ligament, has not been established. In this study, we developed the induction and expansion protocol of LBM cells from human iPSCs, and a high-throughput drug screening system using human LBM cells differentiated from iPSC which is derived from patients with skeletal dysplasia.
[Method] Paired-related homeobox 1 (PRRX1) serves as a marker for the LBM. To assess the induction efficiency of PRRX1 positive cells during each differentiation step, we established PRRX1-tdTomato reporter human iPSC line. Lateral plate mesoderm cells (LPM) derived from PRRX1-tdTomato reporter were used to establish the LBM-inducing method that recapitulates human developmental process. The chondrogenic capacity of expandable LBM (ExpLBM) cells was assessed using our two- or three- dimensional chondrogenic induction method (2DCI or 3DCI). In addition, ExpLBM cells differentiated from iPSCs derived from patients with type II collagenopathy (COL2pathy), one of the skeletal dysplasia arising from mutations in COL2A1, were used to develop high-throughput screening system.
[Results] By activating WNT signaling and inhibiting BMP/TGFb/headge hog signaling pathways, almost all LPM cells could be induced to PRRX1 positive LBM cells. Interestingly, we found the defined culture method that can not only stably expand LBM cells (ExpLBM) but also maintain their PRRX1 expression. ExpLBM formed Alcian Blue positive nodules under 2DCI condition and Safranin O positive cartilaginous particles under 3DCI condition. 2DCI-based high-throughput screening system found that several chemicals improved the chondrogenic capacity of ExpLBM derived from COL2pathy patient.
[Conclusion] ExpLBM cells will be a potential tool to study human bone development, cartilage regeneration and drug discovery using disease-specific human iPSCs.