The primary cilium is organelle that has garnered much attention in the field of cell biology during the last
15 years. It is a slender, solitary hair-like organelle that extends 5-10 uM from each mammalian cell (in the
G0 cell cycle state) that is microtubule-based (9 outer doublets arranged in a circular fashion) and
dependent on a process called Intraflagellar Transport (IFT). IFT is the bidirectional movement of motors
(kinesin-2 in the anterograde and dynein-2 in the retrograde direction) responsible for the assembly and
maintenance of the cilium (Pedersen et. al. 2006). Until this time, it had been labeled a ‘vestigial’ organelle
not worthy of research. Yet, a breakthrough into the sensory role of the primary cilium came in 2000 based
on Dr. Rosenbaum’s research on Chlamydomonas and the motile cilium or flagella. Along with Dr. George
Whitman’s group, they were able to show the importance of Tg737 (IFT88) protein to the pathology of
polycystic kidney disease in mouse (Pazour et al., 2000). Since then, research into the primary cilium has
exploded and has been linked to diverse pathologies (collectively known as ciliopathies) such as retinitis
pigmentosa, hydrocephaly, situs inversus, ovarian and pancreatic cancers among others (Nielsen et al.,
2008; Edberg et al., 2012). Also, various signal transduction pathways have been found to be coordinated
by the primary cilia such as hedgehog, wnt, PDGF among others (Veland et al., 2008).
Thus, in 2006, the Christensen lab at the University of Copenhagen (Denmark) with the collaboration of Dr.
Peter Satir’s group at Albert Einstein College of Medicine (Bronx, NY) began to investigate whether the
human embryonic stem cells possess primary cilium and then to begin preliminary molecular dissections of
the role that this organelle could play in the proliferation and differentiation profiles of these pluripotent
cells. The Albert Einstein group, due to NIH restrictions, had to work with two federally sanctioned cell
lines. Working with the Laboratory of Reproductive Biology at RigsHospital, the Danish side had access to
in-house derived stem cell lines from discarded blastocysts. The advantage for the Danish side was obvious
since these newer cell lines hadn’t undergone as many passages as the NIH-sanctioned lines and were more
robust. To begin preliminary characterizations of these lines, some basic hallmarks of hESCs (Bernhardt et
al., 2012) were found localized to the nucleus as expected such as the transcription factor (TF), Oct4. In
addition, a single primary cilium can be seen denoted by the acetylated tubulin staining emanating from
each cell. Also, the base of the cilium is marked by the presence of pericentrin and centrin which
demarcate the centriole. (Fig. 1)
Fig. 1 Primary cilia stained with anti-acetylated tubulin (tb, red) are indicated by arrows and undifferentiated stem
cells are identified by nuclear colocalization of OCT-4 (green) and DAPI (dark blue) in the merged image (light blue). A
primary cilium (tb, red, arrow) in undifferentiated hESCs emerges from one of the centrioles (asterisks) marked with
anti-centrin (centrin, green). Inset shows anti-pericentrin localization to base of cilia (Pctn, green).
Together, the three labs were the first to discover primary cilia in stem cells while other groups have since
then confirmed these findings (Kiprilov et. al. 2008; Han et. al. 2008). Attention was then to characterize
different signal transduction pathways in the stem cell cilium. Since the hedgehog pathway has been
shown to be important for differentiation and proliferation (Cerdan and Bhatia, 2012), the groups
characterized this signal pathway in these cells using immunofluorescence, electron microscopy and qPCR
techniques. One particularly interesting experiment to show that the hedgehog pathway was functional in
these cells was to add the hedgehog agonist, SAG (Smoothened agonist), and then to isolate the cells for
immunofluorescence at different times. Gradually, one can see the appearance of the smoothened protein
into the cilium as indicated by increasing intensity of the immunofluorescence staining. Conversely,
patched levels in the cilium, decreased. This is a hallmark of hedgehog activation (Fig. 2).
Fig. 2 Immunofluorescence micrographs of hESC showing smo (green), acetylated tubulin (red) and DAPI (blue). The micrographs
from left to right represents SAG treatments at t = 0, 1 and 4 hours.
However, an additional interesting observation was made concerning these stem cells. The hallmark for
stem cells is the presence of certain transcription factors which render these cells in the pluripotent or
undifferentiated state. These include Oct4, Sox2, and Nanog whose localization had been observed in the
Fig. 3 Stem cell markers (Sox2, Nanog, and Oct4) localizing to the nucleus and the primary cilia (arrows) of hESC line LRB003. The
previous 2 figures show shifted overlay images whereby the green and red channels have been slightly shifted such that the red
channel doesn’t swamp out the intensity of the green channels.
nucleus as expected for other TFs. However, the Danish groups curiously found a subpopulation of stem
cells where these TFs were localized to the primary cilium (Fig. 3). This had never been observed or
investigated before. The proper negative controls excluded this from being an artifact (e.g. bleed through).
Thus, it raises an intriguing possibility that perhaps the primary cilia plays a previously uncharacterized role
in the differentiation/proliferation state of the hESCs via possible modifications of these TFs perhaps
analogous to the processing of the Gli transcription factors. Since stem cells are now being more routinely
used for regenerative medicine such as repair of severed spinal cord (Lu e. al. 2012), it behooves us to
better learn the molecular mechanisms that keeps these invaluable cells in an undifferentiated state.
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