Evaluation of in vitro organoid crypt system and contribution of fibroblast-derived Wnt and R spondin to stem cell niche and crypt maintenance

Amie, Charmiah; Pastula, Agnieszka; Powolny, Anna; Quante, Michael

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Title: Evaluation of in vitro organoid crypt system and contribution of fibroblast-derived Wnt and R spondin to stem cell niche and crypt maintenance
Author: Amie, Charmiah
Pastula, Agnieszka
Powolny, Anna
Quante, Michael
Date: 2013
Decade: 2010-2019
Abstract: The goal of this study was to optimize a protocol of murine intestinal crypt 3D culture in preparation for future studies involving 3D co-culture of murine intestinal and cancer cells, and ultimately human 3D tissue cultures. This study was focused on analyzing the role of fibroblasts in regulating epithelial and stem cell physiology and maintenance. Previous studies indicated that Wnt singling pathway was one of the key regulators of the stem cell niche maintenance. Identifying specific signaling ligands contributed by fibroblasts to the stem cell niche can assist in optimizing the 3D culture protocol for future stem cell niche studies. Transgenic mouse models of wild type APC (L2-IL-1b) and APC-modified mice were used as a source of small intestine crypts and fibroblasts. Intestinal crypts were maintained in 3D culture systems and fibroblasts were maintained in classic monolayer. Histological analysis of the 3D cultures revealed presence of crypt like structures which indicated that culture system was effective. RNA isolated from the 3D cultures was used for reverse transcription, and real time-PCR to evaluate expression of genes in the Wnt pathway. Gene expression analysis of normal fibroblasts from normal tissue, Carcinoma Associated Fibroblasts (CAF's) also expressed Rspondins 1, 2, and 3. These results show that fibroblasts indeed express key molecules needed for intestinal stem cell niche maintenance. Identification of the genes contributed by fibroblasts to the stem cell niche physiology will inform further studies examining the influence of stem cell associated fibroblasts on gastrointestinal carcinogenesis.
Document Type: text
Format: application/pdf
Medium: conference proceedings
Collection: Enhancing Global Research and Education in STEM at Spelman College (G-STEM)
Department: Biology
Institution: Spelman College
Geographic Location: Georgia--Atlanta
Handle: http://hdl.handle.net/20.500.12322/sc.gstem:2013_amie_charmiah
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