Generation of human hindlimb/genital tubercle progenitors from pluripotent stem cells

The hindlimbs and genital tubercle arise from a bipotent progenitor population within the posterior lateral plate mesoderm (pLPM) and share common genetic programmes, reflecting their deep evolutionary homology. Perturbations of this shared developmental programme underlie several congenital conditions. Yet most insights into the divergence of pLPM fates come from traditional model organisms, emphasising the need for a human-based system. Here, we report the WNT-FGF-BMP-dependent differentiation of human pluripotent stem cells (hPSCs) into pLPM-derived hindlimb/genital tubercle mesenchymal progenitors (HGTps). We show that BMP signalling plays a pivotal role in driving transcriptomic changes reminiscent of the trunk-to-tail transition—a major reorganisation of the embryonic body plan that initiates pLPM/HGTp specification. We further show that retinoic acid signalling exerts a biphasic effect on LPM specification: early exposure blocks trunk-to-tail transition-like transcriptome changes, whereas late exposure enhances genital tubercle mesenchymal fate by suppressing alternative pLPM derivatives. Strikingly, differentiating genital tubercle mesenchyme self-organises with an overlying epithelium resembling in vivo counterparts. Through xenografting approaches, we show that human HGTp-derived spheroids contribute to the genital tubercle region of the chick embryo revealing their developmental potency. Collectively, our work establishes a platform for the reverse engineering and disease modelling of human HGTps.