Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

Abstract

Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC differentiation, demonstrating the existence of primary cilia and the localization of signaling components in undifferentiated hESCs establishes a mechanistic basis for the regulation of hESC differentiation. Using electron microscopy (EM), immunofluorescence, and confocal microscopies, we show that primary cilia are present in three undifferentiated hESC lines. EM reveals the characteristic 9 + 0 axoneme. The number and length of cilia increase after serum starvation. Important components of the hedgehog (Hh) pathway, including smoothened, patched 1 (Ptc1), and Gli1 and 2, are present in the cilia. Stimulation of the pathway results in the concerted movement of Ptc1 out of, and smoothened into, the primary cilium as well as up-regulation of GLI1 and PTC1. These findings show that hESCs contain primary cilia associated with working Hh machinery.

Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation

“…The A1 peptide tag is specifically recognized by the AcpS enzyme. Enzymatic activity transfers a Ppant group in the CoA substrate to a serine residue in the A1 tag ( (10,(19)(20)(21)(22)(23)(24). Cilial accumulation of A1::Smo::GFP in response to ShhN treatment was confirmed by costaining with two additional primary cilium markers-anti-Arl13b ( Fig.…”

“…17). Multiple components of the Hh pathway localize to the primary cilium or its basal body, including Sonic Hedgehog (Shh) (10,18), Ptch1 (10), Smo (10,(19)(20)(21)(22)(23)(24), suppressor of Fused (suFU) (a key negative regulator of Gli activity), and Gli transcription factors (21,24,25). In the absence of Shh, Ptch1 is enriched in the primary cilium of cultured mammalian cells; its cilial localization is lost after engagement with Shh ligand (10).…”

“…Interestingly, a tryptophan to leucine mutation in the 7th transmembrane helix bundle of Smo generates a dominant-active form (SmoA1), that constitutively localizes to the primary cilium (19,20). Downstream of Smo, suFU and Gli proteins concentrate in or about the primary cilium (21,24,25). Further, their normal function depends on the intact primary cilium (23,(25)(26)(27)(28).…”

Reduced Ciliary Polycystin-2 in Induced Pluripotent Stem Cells from Polycystic Kidney Disease Patients with PKD1 Mutations

“…We next investigated the ability of both PKD and control iPS cells to elaborate primary cilia, which has been previously reported in ES cells. 29 Unstarved, undifferentiated OCT4-positive ES or iPS cells cultured on glass for 7 days in pluripotent stem cell media elaborated short primary cilia containing acetylated a-tubulin ( Figure 3C). Quantitative comparison revealed similar numbers of ciliated cells among ES, control iPS, ADPKD, and ARPKD iPS cells ( Figure 3D).…”

Characterization of primary cilia and Hedgehog signaling during development of the human pancreas and in human pancreatic duct cancer cell lines

“…Recent research has demonstrated that cilia function as sensory units that coordinate a variety of signal transduction pathways essential for embryonic and postnatal development as well as tissue homeostasis in the adult body Eggenschwiler and Anderson, 2007;Kiprilov et al, 2008).…”