Human genetic disorders sharing the common feature of subcutaneous heterotopic ossification (HO) are caused by heterozygous inactivating mutations in GNAS, a gene encoding multiple transcripts including two stimulatory G-proteins, the α-subunit of the stimulatory G-protein (Gsα) of adenylyl cyclase and the ‘extra-long” form of Gsα, XLαs. In one such disorder, progressive osseous heteroplasia (POH), bone formation initiates within subcutaneous fat before progressing to deeper tissues, suggesting that osteogenesis may involve abnormal differentiation of mesenchymal precursors that are present in adipose tissues. We determined by immunohistochemical analysis that GNAS protein expression is limited to Gsα in bone-lining cells and to Gsα and XLαs in osteocytes. By contrast, the GNAS proteins Gsα, XLαs, and NESP55 are detected in adipocytes and in adipose stroma. Although Gnas transcripts, as assessed by qRT-PCR, show no significant changes upon osteoblast differentiation of bone-derived precursor cells, the abundance of these transcripts is enhanced by osteoblast differentiation of adipose-derived mesenchymal progenitors. Using a mouse knockout model, we determined that heterozygous inactivation of Gnas (by disruption of the Gsα-specific exon 1) abrogates upregulation of multiple Gnas transcripts that normally occurs with osteoblast differentiation in wild-type adipose stromal cells. These transcriptional changes in Gnas+/− mice are accompanied by accelerated osteoblast differentiation of adipose stromal cells in vitro. In vivo, altered osteoblast differentiation in Gnas+/− mice manifests as subcutaneous HO by an intramembranous process. Taken together, these data suggest that Gnas is a key regulator of fate decisions in adipose-derived mesenchymal progenitor cells, specifically those that are involved in bone formation.

A microfluidic organotypic device that maintains mouse colon explants for up to 72 h in a physiologically relevant environment is reported. The device is easy to assemble and maintains physiologically accurate oxygen concentrations across the tissue.

It has been shown that short-chain fatty acids (SCFAs) produced by the gut microbiome are of importance to host tissue health; however, measuring such compounds in biological samples is often limited to using hours to days old fecal and blood plasma samples. Organ-on-a-chip models have been created to simplify the complexity but struggle to reproduce the full biology of the gut specifically. We recently reported a tissue-in-a-chip gut model that incorporates gut explanted tissue into a microfluidic device. The system maintains a biologically relevant oxygen gradient and tissue ex vivo for days at a time, but minimal characterization of biological activity was reported. Herein, we use 1 H-NMR to analyze the SCFA content of tissue media effluents from gut explants cultured in the recently developed microfluidic organotypic device (MOD). 1 H-NMR can identify key SCFAs in the complex samples with minimal sample preparation. Our findings show that maintaining physiologically relevant oxygen conditions, something often missing from many other culture systems, significantly impacts the SCFA profile. Additionally, we noted the changes in SCFAs with culture time and potential variability between SCFA levels in male and female mouse tissue explants cultured in the MOD system based on 1 H-NMR spectral profiles.

No abstract