In Vitro Polarization of Colonoids to Create an Intestinal Stem Cell Compartment.
The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture.
Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599 and North Carolina State University, Raleigh, NC, 27695, United States of America.
Peter J
PJ
Attayek
Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599 and North Carolina State University, Raleigh, NC, 27695, United States of America.
Asad A
AA
Ahmad
Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, United States of America.
Yuli
Y
Wang
Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599 and North Carolina State University, Raleigh, NC, 27695, United States of America.
Ian
I
Williamson
Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, United States of America.
Christopher E
CE
Sims
Department of Medicine, Division of Gastroenterology and Hepatology, University of North Carolina, Chapel Hill, NC, 27599, United States of America.
Scott T
ST
Magness
Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599 and North Carolina State University, Raleigh, NC, 27695, United States of America.|Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, United States of America.
Nancy L
NL
Allbritton
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D016764:Cell Polarity; D049109:Cell Proliferation; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology*; D007413:Intestinal Mucosa / Q000166:cytology; D051379:Mice; D008822:Mice, Transgenic; D009940:Organoids / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D019699:Thrombospondins / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism; D060449:Wnt Signaling Pathway; D060509:Wnt3A Protein / Q000378:metabolism
C518903:RSPO1 protein, mouse; D019699:Thrombospondins; D051153:Wnt Proteins; D060509:Wnt3A Protein