Fig. 5: Ectopic ASIP expression in patient-derived iPSCs persists during differentiation.
a, ASIP expression in SVF cell-derived iPSCs from n = 3 clones (as biological independent samples) of the patient and n = 2 clones from each control (Ctr) 1 and 10. b, Immunoblot of ASIP in cell lysates and supernatants from untreated iPSCs from the patient and the two control children. Similar results were obtained by a second independent experiment. c, Persisting ASIP expression in iPSCs of the patient (n = 3 clones as biologically independent samples) but not of control children (n = 2 clones each) differentiated into the three germ layers. Successful differentiation was confirmed by downregulation of the pluripotency markers OCT4, SOX2 and NANOG and upregulation of germ-layer-specific markers (MESP, T and MIXL for mesoderm; PAX6, SOX1 and Nestin for ectoderm; SOX17, FOXA2 and CXCR4 for endoderm). d, Persisting ASIP expression in patient-derived iPSCs (n = 2 clones; red) but not HMGU1 iPSC control cell line (blue) differentiated into hypothalamic-like neurons (HTNs) via neuronal progenitor cells (NPCs). Expression of hypothalamic marker genes LEPR, POMC, AGRP and MC4R confirmed successful differentiation. Gene expression levels were normalized to β-actin and TATA-box-binding protein. ASIP expression is presented as fold change in relation to the mean ± s.e.m. of the controls. Relative expression levels of germ-layer marker genes were calculated by delta-delta Ct method normalized to β-actin. e, Antagonistic activity of ASIP in absence and presence of α-MSH at human MC1R and MC4R. Basal cAMP levels concentration-dependently decreased in presence of increasing concentrations of ASIP (MC1R IC50 = 4.4 nM; MC4R IC50 = 16.9 nM). α-MSH stimulates both MC1R (EC50 = 2.3 nM) and MC4R (EC50 = 10.6 nM). Presence of 100 nM ASIP lowers the potency of α-MSH at MC1R (EC50 = 65 nM) and MC4R (EC50 = 149 nM). Data are mean ± s.e.m. of n = 3 independent experiments each performed in duplicate.
