A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis

Abstract

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.

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation

“…Figure 3). To further verify the enrichment of Gpr182 in CBC ISCs in models without altered Gpr182 expression, the Lgr5-EGFP and Sox9-EGFP BAC-Tg mice were used to isolate different intestinal epithelial cell populations with distinct levels of EGFP, as previously described (18,19,25,(30)(31)(32) Figure 2G). Furthermore, Gpr182 expression was very low in isolated Paneth cells.…”

Dissecting the stem cell niche with organoid models: an engineering-based approach

“…While the capacity to self-organize leads to the formation of reproducible and tightly regulated tissue architectures in vivo, the process can be considerably more variable in vitro, particularly at length scales larger than a few hundred microns (Gracz et al, 2015). Such variability suggests that key chemical, physical and/or spatial cues that guide the progress of self-organization in vivo may be lacking after cell reaggregation in vitro.…”

“…One notable variation on microwells, termed microraft arrays (MRAs; see Glossary, Box 2), utilizes a translucent polystyrene array to facilitate live in-well imaging and controlled release of individual wells for further examination such as gene expression analysis (Gracz et al, 2015;Wang et al, 2010). This method has enabled high-throughput live imaging of intestinal stem cells interacting with other cells in the niche to show that Wnt signaling from Paneth cells is contact dependent (Gracz et al, 2015).…”

“…This method has enabled high-throughput live imaging of intestinal stem cells interacting with other cells in the niche to show that Wnt signaling from Paneth cells is contact dependent (Gracz et al, 2015). Another approach, known as intaglio-void/embed-relief topographic (InVERT) molding (see Glossary, Box 2), uses intaglio/relief-based cell deposition -originally a type of printing technique -to seed one cell population within a recessed surface (e.g.…”

The myeloma stem cell concept, revisited: from phenomenology to operational terms

“…[84][85][86][87] Bmi-1, Notch, Hedgehog, and Wnt, which were initially identified based on their roles in tumor formation, have been shown to be involved in the regulation of self-renewal in normal stem cells in many tissues. [88][89][90] Since myeloma cells are influenced by the host, the microenvironment may play a key role in the initiation of myeloma and associated phenotypic changes-a phenomenon called "plasticity", defined as altered cellular phenotype and function during deregulated differentiation.…”