Single Lgr5 stem cells when co cultured with paneth cells form long lived, self organizing crypt-villus organoids in the absence of non epithelial niche cells. To conclude this fact, stem cells and/or paneth cells were seeded in round bottomed plates in 10% matrigel. Matrigel, a culture medium which provides almost similar environment as likely the complex extracellular environment found in many tissues.
Matrigel based culture system containing,
1. EFG - Epidermal Growth Factors
2. Wnt agonists R-spondin 1 - R-spondin 1 is a secretary protein which interacts with Wnt and activates Wnt signals. (Wnt signals are essential for proliferation).
3. BMP inhibitor Noggin - Noggin is a protein, encoded by NOG gene, binds and inactivates members of signalling proteins BMP (Bone Morphogenetic protein).
Single Lgr5 stem cells autonomously grow into crypt like structures with denovo generated stem cells and paneth cells at their bottom. To conclude that, a confocal cross-sectioning of crypt bottoms of Lgr5 - EGFP - ires - creERT2 mice revealed distribution of paneth cells and Lgr5 stem cells. At the maximum heterotypic contact (paneth cells - stem cells) and minimum of homotypic contact (paneth cells - paneth cells) or (Lgr5 stem cells - Lgr5 stem cells) were found. The same contact was observed in the organoid cultures at crypt bottoms. To get a clear idea about crypt, villus etc... refer following Figure.
Paneth cells and/or stem cells seeded in round bottom plates in 10% Matrigel and following were observed,
GFP organiods formation
a). 500 Lgr5 stem cells - 500 Lgr5 stem cells
(RFP). - 6.7+/- 3.3% of 10 wells / experiment
b). 500 Paneth cells - 500 paneth cells - 0% of 10wells / experiment
(YFP)
c). 500 stem cells - 500 paneth cells - 76.7 +/- 8.8% of 10 wells / experiment
GFP - Green Fluorescent Protein
RFP - Red Fluorescent Protein
YFP - Yellow Fluorescent Protein
Lgr5 stem cells - sorted from a clonal RFP+ organoid culture
Paneth cells - sorted from a clonal YFP+ organoid culture
Thus Lgr5 stem cells and paneth cells require physical contact. Indeed Lgr5 stem cells are critically dependent on Notch signals.
Stem cell markers.
1. Notch 1
2. Olfm4
3. FZD7
4. Cdca7
5. Tnfrsf19
6. Lgr5
Paneth cell Markers
1. Dll4
2. Tgfa
3. Egf
4.Wnt11
5. Wnt3
6. Defa1
7. Lyz1
Among genes, most highly enriched in paneth cells, we noted Wnt3, Wn11, Egf, Tgfa and notch ligand Dll4. Thus paneth cells provide essential signals for stem cells support. High level expression of Wnt3 was confirmed by in situ hybridization. Indeed, Wnt signalling instructs intestinal cells to adopt a proliferative progenitor phenotype. So, when an Wnt secretion inhibitor (porcupine inhibitor) Iwp1 was added, Organoid was entirely lost and proliferation halted. It can be overcome by exogenous Wnt3A. It was concluded that R spondin 1 acts by amplifying the local response to short - range Wnt produced by paneth cells. Thus only the direct neighbours of paneth cells, the Lgr5 stem cells receive strong Wnt signals which can be further, increased by R-spondin 1.
It was recently observed that stem cells and paneth cells doublets strongly increased plating efficiency compared to single stem cells. To investigate that concept using in vivo models, whether paneth cells provide essential support to Lgr5 stem cells, used 3 genetic mouse models for paneth cell loss.
A. Mutation of Gfi1
B.Transgenic expression of diphtheria toxinA4 under the paneth cells - specific cryptdin 2 promoter (CR2 - tox176)
C. Conditional deletion of Sox 9
In crypts of Gfi 1 and CR 2 - tox176 (A and B) paneth cells were seen in decreased number. Number of stem cells were again decreased, coincident with paneth cells.
Thus paneth cells serve as multifunctional guardians of stem cells both by secreting bactericidal products and by providing essential niche signals for organoid formation.
Reference: http://www.ncbi.nlm.nih.gov/pubmed/21113151
Picture Reference : nature
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