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Engineering transplantable jejunal mucosal grafts using primary patient-derived organoids for children with intestinal failure

ABSTRACT: Intestinal failure (IF), following extensive anatomical or functional loss of small intestine (SI), hasdebilitating long-term consequences on children1. Priority of care is to increase the child’s length of functional intestine, jejunum in particular, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using primary patient biomaterials. We show that organoids derived from patients can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which form optimal biological scaffolds. Remarkably, proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human SI and colon scaffolds, indicating that both can be used interchangeably as platforms for intestinal engineering. Indeed, seeding jejunal organoids onto either scaffold type reliably reconstructs grafts that exhibit several aspects of physiological jejunal function with potential to survive after transplantation. Our findings provide proof-of-concept data for engineering IF patient-specific jejunal grafts, ultimately aiding in restoration of nutritional autonomy.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colon, Small Intestine

SUBMITTER: Peter Faull

LAB HEAD: Bram Snijders

PROVIDER: PXD019816 | Pride | 2020-09-16

REPOSITORIES: Pride

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			Action	DRS
	DGL8846A10RW3-40-550198.raw	Raw
	DGL8846A11RW3-40-550193.raw	Raw
	DGL8846A12RW3-40-550194.raw	Raw
	DGL8846A13RW3-40-550195.raw	Raw
	DGL8846A14RW3-40-550196.raw	Raw

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Project description:The endodermal lining of the adult gastro-intestinal tract harbors stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation program and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. Conversely, forced expression of Cdx2 in gastric organoids results in their intestinalization. The intestinal genetic program is thus critically dependent on the single transcription factor encoding gene Cdx2. Small intestinal crypts and stomach glands were isolated from Cdx2-/fl / Lgr5-EGFP-CreERT2 mice and cultured for a week in order to generate small intestinal (SI) and stomach (Sto) in vitro organoids. The Lgr5-CreERT2 enzyme activity has been induced by overnight 4-hydroxytamoxifen induction. Tamoxifen treated and untreated Lgr5-EGFPhi SI and Sto stem cells were FACS sorted and seeded back into ENRWfg (Sto med) culture conditions in order to generate Cdx2-/fl small intestinal (Control SI), Cdx2null small intestinal (Cdx2null SI) and Cdx2-/fl stomach (Control Sto) clonal organoids. Cdx2-/fl SI organoids and Cdx2-/fl Sto organoids have been also cultured in ENR (SI med) to induce differentiation. After some passages of clonal organoid expansion, RNA was isolated from Control SI, Cdx2null SI and Control Sto Lgr5-EGFPhi FACS sorted stem cell populations and from smal intestinal and stomach organoids cultured in different conditions and hybridized on Affymetrix Mouse Gene ST 1.1 arrays.

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Engineering transplantable jejunal mucosal grafts using primary patient-derived organoids for children with intestinal failure

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