BACKGROUND & AIMS Reserve intestinal stem cells (rISCs) are quiescent/slowly cycling under homeostatic conditions, allowing for their identification with label-retention assays. rISCs mediate epithelial regeneration after tissue damage by converting to actively proliferating stem cells (aISCs) that self renew and demonstrate multipotency, which are defining properties of stem cells. Little is known about the genetic mechanisms that regulate the production and maintenance of rISCs. High expression levels of the transcription factor Sox9 (Sox9high) are associated with rISCs. This study investigates the role of SOX9 in regulating the rISC state. METHODS We used fluorescence-activated cell sorting to isolate cells defined as aISCs (Lgr5high) and rISCs (Sox9high) from Lgr5EGFP and Sox9EGFP reporter mice. Expression of additional markers associated with active and reserve ISCs were assessed in Lgr5high and Sox9high populations by single-cell gene expression analyses. We used label-retention assays to identify whether Sox9high cells were label-retatining cells (LRCs). Lineage-tracing experiments were performed in Sox9-CreERT2 mice to measure the stem cell capacities and radioresistance of Sox9-expressing cells. Conditional SOX9 knockout mice and inducible-conditional SOX9 knockout mice were used to determine whether SOX9 was required to maintain LRCs and rISC function. RESULTS Lgr5high and a subset of crypt-based Sox9high cells co-express markers of aISC and rISC (Lgr5. Bmi1. Lrig1, and Hopx). LRCs express high levels of Sox9 and are lost in SOX9-knockout mice. SOX9 is required for epithelial regeneration after high-dose irradiation. Crypts from SOX9-knockout mice have increased sensitivity to radiation, compared with control mice, which could not be attributed to impaired cell-cycle arrest or DNA repair. CONCLUSIONS SOX9 limits proliferation in LRCs and imparts radiation resistance to rISCs in mice.

Stem cells reside in “niches”, where support cells provide signaling critical for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a “microraft array” (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells co-cultures. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for qPCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.

Abstract:The catastrophic 8.0 Richter magnitude earthquake that occurred on 12 May 2008 in Wenchuan, China caused extensive damage to vegetation due to widespread landslides and debris flows. In the past five years, the Chinese government has implemented a series of measures to restore the vegetation in the severely afflicted area. How is the vegetation recovering? It is necessary and important to evaluate the vegetation recovery effect in earthquake-stricken areas. Based on MODIS NDVI data from 2005 to 2013, the vegetation damage area was extracted by the quantified threshold detection method. The vegetation recovery rate after five years following the earthquake was evaluated with respect to counties, altitude, fault zones, earthquake intensity, soil texture and vegetation types, and assessed over time. We have proposed a new method to obtain the threshold with vegetation damage quantitatively, and have concluded that: (1) The threshold with vegetation damage was 13.47%, and 62.09% of the field points were located in the extracted damaged area; (2) The total vegetation damage area was 475,688 ha, which accounts for 14.34% of the study area and was primarily distributed in the central fault zone, the southwest mountainous areas and along rivers in the Midwest region of the study area; (3) Vegetation recovery in the OPEN ACCESS Remote Sens. 2015, 7 8758 damaged area was better in the northeast regions of the study area, and in the western portion of the Wenchuan-Maoxian fracture; vegetation recovery was better with increasing altitude; there is no obvious relationship between clay content in the topsoil and vegetation recovery; (4) Meadows recovered best and the worst recovery was in mixed coniferous broad-leaved forest; (5) 81,338 ha of vegetation in the damage area is currently undergoing degradation and the main vegetation types in the degradation area are coniferous forest (31.39%) and scrub (34.17%); (6) From 2009 to 2013, 41% has been restored to the level before the earthquake, 9% has not returned but 50% will continue to recover. The Chinese government usually requires five years as a period for post-disaster reconstruction. This paper could be regarded as a guidance for Chinese government departments, whereby additional investment is encouraged for vegetation recovery.

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