Abstract
Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.
Highlights
The non-motile primary cilium is an organelle composed of tubulin, which projects from the centrosome and is located at the apical cell surface
Cilia length tended to vary with phenotype: cells grown in Human adipose-derived stem cells (hASC) growth media tended to express shorter cilia (Fig. 1b, 1c, 1e), while osteogenically stimulated hASC expressed more extended cilia (Fig. 1c, 1f) as they committed to the osteogenic lineage, with the day 12 osteogenically differentiated hASC expressing the longest cilia (Fig. 1f)
Evaluation of osteopontin gene expression was assessed at day 15 of osteogenic differentiation; there was no significant change in osteopontin expression with knockdown. These results suggest that PC1 and PC2 may function in intermediate signaling processes related to hASC osteogenesis, intraflagellar transport protein-88 (IFT88) function is necessary at the early-to-intermediary duration of the osteogenic differentiation process, and PC1 in particular is critical in later steps of the osteogenic differentiation process
Summary
The non-motile primary cilium is an organelle composed of tubulin, which projects from the centrosome and is located at the apical cell surface. Discovered over a century ago, non-motile primary cilia were largely considered vestigial organelles, despite their prevalence on a variety of cell types [1]. They have been implicated as critically important chemo- and mechanoresponsive cell surface structures, hinting at their role in functional phenotypic maintenance in a variety of mammalian cell types [2,3]. The scope of the primary cilium’s function remains largely elusive, though evidence suggests that its function is complex It acts as an important site for intracellular signaling [4] and detects external chemical and mechanical changes in the extracellular environment [5]. Emerging evidence from our group and others suggests that, in addition to tissue homeostasis, they may be involved in signaling stem cell lineage commitment in both embryonic as well as in adult stem cells, both in vivo and in vitro [5,6,7]
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