Distinct Levels of Sox9 Expression Mark Colon Epithelial Stem Cells that Form Colonoids in Culture

Sox9 is an high-mobility group box transcription factor that is expressed in the stem cell zone of the small intestine and colon. We have previously used a Sox9EGFP mouse model to demonstrate that discrete levels of Sox9 expression mark small intestine epithelial stem cells that form crypt/villus-like structures in a three-dimensional culture system (Formeister EJ, Sionas AL, Lorance DK, Barkley CL, Lee GH, Magness ST. Am J Physiol Gastrointest Liver Physiol 296: G1108–G1118, 2009; Gracz AD, Ramalingam S, Magness ST. Am J Physiol Gastrointest Liver Physiol 298: G590–G600, 2010). In the present study, we hypothesized that discrete levels of Sox9 expression would also mark colonic epithelial stem cells (CESCs). Using the Sox9EGFP mouse model, we show that lower levels of Sox9 mark cells in the transit-amplifying progenitor cell zone, while higher levels of Sox9 mark cells in the colonic crypt base. Furthermore, we demonstrate that variable SOX9 levels persist in cells of colonic adenomas from mice and humans. Cells expressing lower Sox9 levels demonstrate gene expression profiles consistent with more differentiated populations, and cells expressing higher Sox9 levels are consistent with less differentiated populations. When placed in culture, cells expressing the highest levels of Sox9 formed “colonoids,” which are defined as bodies of cultured colonic epithelial cells that possess multiple cryptlike structures and a pseudolumen. Cells expressing the highest levels of Sox9 also demonstrate multipotency and self-renewal in vitro, indicating functional stemness. These data suggest a dose-dependent role for Sox9 in normal CESCs and cells comprising colon tumors. Furthermore, distinct Sox9 levels represent a new biomarker to study CESC and progenitor biology in physiological and disease states

Assessing the sensitivity and repeatability of permanganate oxidizable carbon as a soil health metric: An interlab comparison across soils

Soil organic matter is central to the soil health framework. Therefore, reliable indicators of changes in soil organic matter are essential to inform land management decisions. Permanganate oxidizable carbon (POXC), an emerging soil health indicator, has shown promise for being sensitive to soil management. However, strict standardization is required for widespread implementation in research and commercial contexts. Here, we used 36 soils—three from each of the 12 USDA soil orders—to determine the effects of sieve size and soil mass of analysis on POXC results. Using replicated measurements across 12 labs in the US and the EU (n = 7951 samples), we quantified the relative importance of 1) variation between labs, 2) variation within labs, 3) effect soil mass, and 4) effect of soil sieve size on the repeatability of POXC. We found a wide range of overall variability in POXC values across labs (0.03 to 171.8%; mean = 13.4%), and much of this variability was attributable to within-lab variation (median = 6.5%) independently of soil mass or sieve size. Greater soil mass (2.5 g) decreased absolute POXC values by a mean of 177 mg kg−1 soil and decreased analytical variability by 6.5%. For soils with organic carbon (SOC) >10%, greater soil mass (2.5 g) resulted in more frequent POXC values above the limit of detection whereas the lower soil mass (0.75 g) resulted in POXC values below the limit of detection for SOC contents −1 while decreasing the analytical variability by 1.8%. In general, soils with greater SOC contents had lower analytical variability. These results point to potential standardizations of the POXC protocol that can decrease the variability of the metric. We recommend that the POXC protocol be standardized to use 2.5 g for soils <10% SOC. Sieve size was a relatively small contributor to analytical variability and therefore we recommend that this decision be tailored to the study purpose. Tradeoffs associated with these standardizations can be mitigated, ultimately providing guidance on how to standardize POXC for routine analysis.</p