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RNA-sequencing of skeletal stem cells (SSC) and mesenchymal stem cells (MSC) from hindlimb unloaded and reloaded mice

ABSTRACT: Disuse osteoporosis (DO) occurs in response to mechanical unloading of the skeleton and results in structural changes that compromise bone strength leading to increased fracture risk. Although skeletal stem cells (SSCs) are the main source of osteoblasts and adipocytes in adult bone marrow, whether DO impacts SSC function has not been established. Here, three-month-old male C57BL/6 mice were subjected to a hindlimb unloading (HU) model of disuse osteoporosis for 8 or 14 weeks by tail suspension. Control mice (ambulatory or AMB) were allowed normal use of their hindlimbs. An additional cohort underwent an 8-week recovery period after 8-weeks of unloading. Bone marrow-derived, LepR+ skeletal stem cells (SSCs) were isolated by FACS (n=3 or 4) and pooled prior to RNA isolation. Where indicated, cultured, immuno-depleted, bone marrow-derived mesenchymal stem cells (MSCs) were also collected (n=3) and pooled prior to RNA isolation. Generated libraries were subjected to NGS RNA-seq analysis on the Illumina NextSeq 500. These data delineate unique physiological adaptations to prolonged unloading, demonstrate that DO results in increased leptin expression and sympathetic signaling within the BM niche.

ORGANISM(S): Mus musculus

PROVIDER: GSE156174 | GEO | 2021/12/01

REPOSITORIES: GEO

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Project description:Prolonged skeletal unloading through bedrest results in bone loss similar to that observed in elderly osteoporotic patients, but with an accelerated timeframe. This rapid effect on weight-bearing bones is also observed in astronauts who lose up to 2% of their bone mass per month spent in Space. Despite important implications for Spaceflight travellers and bedridden patients on Earth, the exact mechanisms involved in disuse osteoporosis have not been elucidated. Parathyroid hormone-related protein (PTHrP) regulates many physiological processes including skeletal development, and has been proposed as a gravisensor. To investigate the role of PTHrP in microgravity-induced bone loss, trabecular osteoblasts (TOs) from Pthrp+/+ and -/- mice were exposed to simulated microgravity for 6 days. Viability of TOs decreased in inverse proportion to PTHrP expression levels. Microarray analysis of Pthrp+/+ TOs after 6 days at 0g revealed expression changes in genes encoding prolactins,apoptosis and survival molecules, bone metabolism and extra-cellular matrix composition proteins, chemokines, IGF family and Wnt-related signalling molecules. Importantly, 88% of 0g-induced expression changes in Pthrp+/+ cells overlap those observed in Pthrp-/- cells in normal gravity. Pulsatile treatment with PTHrP1-36 peptide during microgravity exposure reversed a large proportion of 0g-induced changes in Pthrp+/+ TOs. Our results confirm PTHrP efficacy as an anabolic agent to prevent microgravity-induced cell death in TOs. Total RNA samples extracted from Pthrp+/+and -/- trabecular osteoblasts (TOs) exposed for 6 days to simulated 0g in Synthecon rotating cell, or left 6 days in culture at 1g. Cells had either been treated with a pulsatile treatment (2 h/day) of PTHrP1-36 peptide (10-8M) or received a change in growth medium. In total: 8 different conditions with 2 replicates each, i.e. Pthrp+/+ TOs at 0g or 1g with or without PTHrP1-36 treatment, and Pthrp-/- TOs at 0g or 1 g,with or without PTHrP1-36 treatment.

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RNA-sequencing of skeletal stem cells (SSC) and mesenchymal stem cells (MSC) from hindlimb unloaded and reloaded mice

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