Primary Cilia Are Essential For Maintaining Bone Marrow Stromal Cells In Their In Situ Environment

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1 Primary Cilia Are Essential For Maintaining Bone Marrow Stromal Cells In Their In Situ Environment Thomas R. Coughlin 1, Matthew G. Haugh, B.A., B.A.I., PhD 2, Muriel Voisin 3, Evelyn Birmingham 4, Laoise M. McNamara 4, Glen L. Niebur 1. 1 University of Notre Dame, Notre Dame, IN, USA, 2 National University of Ireland Galway, Galway, Ireland, 3 NationalUniversity of Ireland, Galway, Galway, Ireland, 4 National University of Ireland, Galway, Galway, Ireland. Disclosures: T.R. Coughlin: None. M.G. Haugh: None. M. Voisin: None. E. Birmingham: None. L.M. McNamara: None. G.L. Niebur: None. Introduction: Mesenchymal stem cells (MSCs) are multipotent cells that reside in the bone marrow and differentiate into connective tissue cells, such as adipocytes and osteoblasts [1]. Maintenance, proliferation, and osteogenic differentiation of MSCs are essential to skeletal health. Differentiation of MSCs is influenced by mechanical stimuli in vitro [2]. in vivo, low magnitude mechanical signals (LMMS) increased MSC number by 46% and the differentiation capacity of MSCs was biased towards osteoblastic differentiation rather than adipogenesis [3]. Thus, mechanobiological stimuli may play an important role in maintaining balanced MSC differentiation. Primary cilia are present on MSCs, osteoblasts, and osteocytes when cultured in 2-D, and fluid flow stimulation of such cells leads to production of osteogenic factors [4]. Primary cilia knockout in osteoblasts and osteocytes in a mouse model decreased the bone formation and mineral apposition rates [6]. in vitro, when primary cilia were inhibited using sirna in MSCs, there was a 2.8-fold increase in the MSC proliferation rate in response to flow compared to controls [5]. However, these 2-D in vitro studies do not represent the in vivo marrow environment, and, as such the role of primary cilia on MSCs in situ is not fully understood. The goal of this study was to determine the effect of primary cilia knockdown on the population of stromal cells in 3-D, ex vivo culture, both with and without mechanical loading. Specifically, we used a vibrational bioreactor to culture trabecular bone explants, knocked down the primary cilia with chloral hydrate, and quantified the effect on stromal cell population number and proliferative potential for both static and mechanically stimulated (LMMS) constructs. Methods: Trabecular bone explants were obtained from the cervical vertebrae of young sheep, 6 to 10 months of age. The end plates of the C2 vertebra were removed, and 10 mm diameter cylindrical explants were obtained using a diamond core drill. The explants were placed into a bioreactor that supplied a continuous flow of media (88% HG DMEM, 10% FBS, 2% antibiotics) and simultaneously applied LMMS to the explants. To verify primary cilia presence and knockdown, six explants from three sheep were cultured for 1 week. After 48 h in culture, three explants were treated to knockdown the primary cilia by addition of 4 mm chloral hydrate (CH) to the media for 72 h, and then given standard media for 48 h. The remaining three explants were cultured for 1 wk with standard media. At 1 wk, explants were fixed, demineralized, paraffin embedded, sectioned at 20 μm, and immunohistochemistry (IHC) was performed using antiacetylated α-tubulin (Abcam 6-11B-1) to target the primary cilium (two sections per explant). The primary cilia antibodies were verified using a positive control. Ovine kidney sections were immunostained using two established primary cilia antibodies, acetylated α-tubulin and intraflagellar transport protein 88. Three image stacks were obtained per section using an inverted confocal laser-scanning microscope (Nikon) at 63x magnification with a 7 s scan speed, averaging 4 images. Primary cilia were identified based on two criteria: being proximal to a cell nucleus (PI), and taining of at least 1 μm in length. The number of ciliated cells was normalized to the total number of cells in the imaged volume, which was determined using the Automatic Nuclei Counter plugin for ImageJ (NIH). To assess the effect of LMMS on the stromal population, 12 explants were obtained from four sheep. Four groups of three explants from different sheep were studied: control (CNT) and LMMS, with and without CH (±CH). CH was applied to +CH groups on days 2 through 5 and 10 throug 13. On days 5 to 10 and 13 to 18, LMMS groups were subjected to 30 Hz, 0.3 g vibration for 1 h a day. After 19 days in culture (7 days after the final CH treatment) marrow was removed from the explants through three steps of trypsinization and centrifugation. Colony forming unit (CFU) assays were performed in triplicate on each explant, with the exception of +CH groups, which did not have enough cells for triplicate. Harvested cells were plated at a density of 100,000 cells/well in six-well plates, and cultured for

2 5 days, after which cells were fixed and stained with crystal violet. Colonies were counted manually and measured for area using ImageJ (NIH). Results: The bone marrow maintained a normal morphology following 19 d in culture (Fig. 1A). Primary cilia projected into the intercellular space (Fig. 1B). Primary cilia incidence in bone marrow cells increased after 1 wk of bioreactor culture compared to day 0 (Fig. 1C; p<0.05). After 19 d in culture, an average of 1.5 and 0.5 million cells were isolated from the marrow in the trabecular bone explants, in the -CH and +CH explants, respectively. There were seven times more harvested cells formed colonies in the -CH groups compared to +CH groups (Fig. 2A; p<0.005), while LMMS did not affect colony number (Fig. 2A). However, colonies were larger in the LMMS group compared to the CNT group, indicating a higher proliferative potential (Fig. 2B; p<0.05). This effect was lost in the +CH group. Discussion: Primary cilia knockdown in bone marrow negatively affected the stromal cell fraction in this 3-D explant model. These results indicate that cilia play an important role in maintaining the marrow cell population. While LMMS did not affect the stromal cell population number, it did increase their proliferative potential. This effect was diminished after primary cilia knockdown. Taken together, primary cilia appear to have both mechanosensory and non-mechanosensory roles in maintaining the marrow cell population. The ex vivo culture methodology developed in this study provided a unique means to examine the effects of CH on the stromal cell population within bone marrow. Understanding the differences between the behavior of cells in 2-D vs. 3-D culture is essential to translating in vitro results to in vivo situations. Also, the explant model provides more realistic scenarios for mechanical stimulation of bone marrow cells. Our data showed decreased MSC populations in explants, while a previous study using 2-D study showed MSC proliferation increased in response to flow when primary cilia were knocked down [5]. In the bioreactor system, cells are not directly exposed to flow, but may experience shear stress from cell-cell contact inducing an altered response. CH disrupts microtubules, affecting proliferation [7]. This may explain the decreased cell populations in the +CH groups. Although, CH did not decrease the relative number of primary cilia in our explants, in previous studies primary cilia present after 20 h of CH treatment were distorted and did not respond to fluid flow with an increase in Ca 2+, while untreated cells did [8]. Similarly, in this study, the number of cells presenting primary cilia knockdown may not have decreased, but the resulting mechano-sensitivity was affected. In the current study primary cilia were found to be essential for maintaining the marrow cell population and their increase in proliferative potential in response to loading. Significance: The results of this ex vivo culture study provide evidence that the primary cilium plays a role in maintaining MSC proliferation, and thereby shedding light into their in vivo role. Acknowledgments: SFI 07/EN/E015B-ISTTF 11; ERC Grant no (BONEMECHBIO); NSF CMMI References: 1. Gimble (1996) Bone 19(5). 2. Riddle (2006) Am J Physiol Cell Physiol. 3. Luu (2009)JBMR 24(1). 4. Hoey (2012) Front Endocrinol. 5. Hoey (2012) Stem Cells 30(11). 6. Temiyasathit (2012) PLoS One 7(3). 7. Lee (1987) Journal of Cell Science Praetorius and Spring (2002) J Membrane Biol 191.

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4 ORS 2014 Annual Meeting Poster No: 0578

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