Cell-based RNAi screening and high-content analysis in primary calvarian osteoblasts applied to identification of osteoblast differentiation regulators

Abstract

Osteoblasts are responsible for the maintenance of bone homeostasis. Deregulation of their differentiation is etiologically linked to several bone disorders, making this process an important target for therapeutic intervention. Systemic identification of osteoblast regulators has been hampered by the unavailability of physiologically relevant in vitro systems suitable for efficient RNAi and for differentiation read-outs compatible with fluorescent microscopy-based high-content analysis (HCA). Here, we report a new method for identification of osteoblast differentiation regulators by combining siRNA transfection in physiologically relevant cells with high-throughput screening (HTS). Primary mouse calvarial osteoblasts were seeded in 384-well format and reverse transfected with siRNAs and their cell number and differentiation was assayed by HCA. Automated image acquisition allowed high-throughput analyses and classification of single cell features. The physiological relevance, reproducibility, and sensitivity of the method were validated using known regulators of osteoblast differentiation. The application of HCA to siRNAs against expression of 320 genes led to the identification of five potential suppressors and 60 activators of early osteoblast differentiation. The described method and the associated analysis pipeline are not restricted to RNAi-based screening, but can be adapted to large-scale drug HTS or to small-scale targeted experiments, to identify new critical factors important for early osteoblastogenesis.

A variant in IL6ST with a selective IL-11 signaling defect in human and mouse

“…35 Interestingly, suture fusion occurred in mice with incomplete depletion, pinpointing to a certain threshold of MSC number that is needed to maintain suture patency. IL-11 promotes osteoblast differentiation into osteogenic cells in vitro [36][37][38] and supports osteoclastogenesis through induction of TNFSF11 (previously termed as RANKL) expression in osteoblasts and also effects on osteoclasts. 6,39 It is therefore tempting to speculate that IL-11 signaling is responsible for an increase in craniofacial MSC turnover but is not necessary for MSC maintenance.…”

Inhibition of Cdk5 Ameliorates Skeletal Bone Loss in Glucocorticoid-Treated Mice

“…Primary calvarial osteoblast isolation was performed using neonatal mouse calvaria of 2–5-day-old pups as previously described [ 54 , 55 ]. Briefly, the calvariae were isolated in 1 mL digestion solution (0.2% w / v each of collagenase A and dispase II (Roche, Basel, Switzerland)) and incubated at 37 °C for 10 min at 700 rpm on a shaker.…”

“…Following overnight incubation (37 °C; 5% CO 2 ), the medium was replaced with a fresh medium. The experiments were performed at an 80% confluency as previously described [ 53 , 54 ].…”

“…SMARTpool non-targeting siRNA control (si NT ) and Cdk5 -specific siRNA (si Cdk5 ) were purchased from Horizon Discovery (Waterbeach, UK). The transfection was performed using a final concentration of 20 nM siRNA with 0.125% Lipofectamine RNAiMAX transfection reagent (Life Technologies, ThermoFisher Scientific, Waltham, MA, USA), as previously described [ 54 ]. The siRNA sequences used are listed in Table 1 .…”

“…For quantitative Alp, primary murine calvarial osteoblasts were reversely transfected and seeded in a 384-well plate. The cells were differentiated by adding an osteogenic induction medium, fixed and stained with ELF 97 (Thermo Fisher Scientific, Waltham, MA, USA) for Alp, and DRAQ5 (BioStatus Ltd., Loughborough, UK) for nuclei, and were analyzed as previously described [ 54 ]. For Cdk5 inhibition in vitro, primary murine calvarial osteoblasts were treated with either a vehicle or roscovitine (0.16 µM) for six days as previously described [ 53 ].…”