A Proximal-to-Distal Survey of Healthy Adult Human Small Intestine and Colon Epithelium by Single-Cell Transcriptomics

Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells

“…Transfecting ribonucleoprotein (RNP) complexes instead of Cas9-expressing plasmids and guide RNAs (gRNAs) independently is attractive for delivering CRISPR reagents owing to the decreased potential for off-target cleavage or integration ( Liang et al., 2015 ). As a candidate locus to test RNP targeting efficiency in hISCs, OLFM4 was chosen for its robust expression in small intestinal hISCs, as demonstrated by in situ hybridization, immunostaining, and single-cell RNA sequencing ( Burclaff and Bliton, 2022 ; Busslinger et al., 2021 ; van der Flier et al., 2009 ; Gersemann et al., 2012 ).…”

“…In this study we generated and characterized an OLFM4-emGFP hISC line. OLFM4 has been shown by in situ hybridization and scRNA sequencing to be robustly expressed by human crypt-base columnar cells (CBCs), with gradually decreasing expression up the crypt axis ( Burclaff and Bliton, 2022 ; Busslinger et al., 2021 ; van der Flier et al., 2009 ). While OLFM4 demonstrates broader crypt expression than LGR5 , single-cell RNAseq data show that the highest OLFM4 expression overlaps with the highest LGR5 expression ( Burclaff and Bliton, 2022 ).…”

“…OLFM4 has been shown by in situ hybridization and scRNA sequencing to be robustly expressed by human crypt-base columnar cells (CBCs), with gradually decreasing expression up the crypt axis ( Burclaff and Bliton, 2022 ; Busslinger et al., 2021 ; van der Flier et al., 2009 ). While OLFM4 demonstrates broader crypt expression than LGR5 , single-cell RNAseq data show that the highest OLFM4 expression overlaps with the highest LGR5 expression ( Burclaff and Bliton, 2022 ). Using engineered gut epithelial tissue constructs generated on planar-crypt microarrays (PCMs), our data revealed a robust gradient of OLFM4-emGFP expression levels with a direct correlation with endogenous OLFM4 , LGR5 , and KI67 levels, and highest hISC organoid forming capacity correlated with highest OLFM4-emGFP expression and smaller cell diameter.…”

Expression Microdissection for use in qPCR based analysis of miRNA in a single cell type

“…The optimized xMD method, described here, can allow any researcher access to in vivo miRNA expression patterns from cells of interest from FFPE tissues. Between single cell human cell atlas projects and the human protein atlas, a large number of cell-type defining genes/proteins are being realized that can further refine the selectivity of this xMD method (cite single cell papers 8,21,22,26,27 . Additionally, most materials used in this optimized version of xMD are widely available and cost-effective.…”

“…21,22,26,27 . Additionally, most materials used in this optimized version of xMD are widely available and cost-effective.…”