Generation of human hypothalamic appetite-regulating neurons and tanycytes from stem cells

The balance between energy intake and expenditure is controlled by the hypothalamus, a small brain region composed of several subregionalised nuclei with high neuronal diversity which are key to controlling appetite and food intake through response to circulating hormones and metabolic. However, the cellular and functional characteristics of this highly specialised neural region has been studied mainly in animal models due to a lack of access to human experimental counterparts. Here, we fine-tuned the differentiation of human pluripotent stem cells toward hypothalamic nuclei, including the arcuate nucleus (ARC), ventromedial hypothalamus (VMH) and paraventricular nucleus (PVN), and we identified key subtype-specific progenitor markers of hypothalamic subregions. We show that the timing for addition and withdrawal of bone morphogenic protein (BMP) was essential for controlling sub-regional specification of tuberal hypothalamic progenitors along the anterior-posterior axis, balancing VMH versus ARC fates. A particular population of SHH-/NKX2.1+/RAX+/FGF10+/TBX3high posterior tuberal progenitors was identified as the source for generation of ARC-associated agouti-related peptide (AGRP) neurons and tanycytes whilst anterior tuberal SHH+/NKX2.1+/RAX+/TBX3low progenitors generated VMH phenotypes including NR5A1 neurons. Upon maturation in 2D, 3D and in vivo, ARC-patterned progenitors gave rise to multiple key appetite-regulating cell types including AGRP, prepronociceptin (PNOC), growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH) and pro-opiomelanocortin (POMC) neurons and characteristic tanycyte glial cells. Differentiated ARC cultures showed high transcriptomic similarity to the human ARC and displayed evidence of functionality by AGRP secretion and responsiveness to leptin and fibroblast growth factor 1 (FGF1). In summary, our work provides new insights into the developmental lineages underlying human hypothalamic subregional specification and enables access to highly characterized human hypothalamic ARC cultures, which will provide novel opportunities for investigating the cellular and molecular pathways triggered by obesity-associated genetic variants and appetite-regulating drugs and peptides.