Limits...
Intestinal Commitment and Maturation of Human Pluripotent Stem Cells Is Independent of Exogenous FGF4 and R-spondin1.

Tamminen K, Balboa D, Toivonen S, Pakarinen MP, Wiener Z, Alitalo K, Otonkoski T - PLoS ONE (2015)

Bottom Line: The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids.FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage.Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro.

View Article: PubMed Central - PubMed

Affiliation: Research Programs Unit, Molecular Neurology and Biomedicum Stem Cell Centre, University of Helsinki, Helsinki, Finland.

ABSTRACT
Wnt/beta-catenin signaling plays a central role in guiding the differentiation of the posterior parts of the primitive gut tube into intestinal structures in vivo and some studies suggest that FGF4 is another crucial factor for intestinal development. The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids. The most prominent induction of the well-established intestinal marker gene CDX2 was achieved when hPSC-derived definitive endoderm cells were treated with Wnt agonist molecule CHIR99021 during differentiation to hindgut. FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage. When hindgut stage cells were further cultured in 3D, they formed self-renewing organoid structures containing all major intestinal cell types even without exogenous R-spondin1 (RSPO1), a crucial factor for the culture of epithelial organoids derived from adult intestine. This may be explained by the presence of a mesenchymal compartment in the hPSC-derived organoids. Addition of WNT3A increased the expression of the Paneth cell marker Lysozyme in hPSC-derived organoid cultures, whereas FGF4 inhibited both the formation and maturation of intestinal-like organoids. Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro.

No MeSH data available.


Related in: MedlinePlus

HPSC-derived organoids express intestinal marker genes at levels comparable to human intestinal epithelium.Results represent day 33 organoids (d42 from hPSC) derived from day 9 CHIR cells. A. Gene expression of intestinal differentiation markers CDX2, KRT20, KLF5, IFABP2 and HOXA13. B. Expression of crypt area/intestinal stem cell markers LGR5, ASCL2 and SOX9. Data represent the mean ± SEM of 3–9 samples. (E, EGF; N, Noggin; R, R-Spondin1; W, WNT3A; F, FGF4; 2D, initial monolayer culture; HI, human intestinal epithelium samples.)
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4521699&req=5

pone.0134551.g005: HPSC-derived organoids express intestinal marker genes at levels comparable to human intestinal epithelium.Results represent day 33 organoids (d42 from hPSC) derived from day 9 CHIR cells. A. Gene expression of intestinal differentiation markers CDX2, KRT20, KLF5, IFABP2 and HOXA13. B. Expression of crypt area/intestinal stem cell markers LGR5, ASCL2 and SOX9. Data represent the mean ± SEM of 3–9 samples. (E, EGF; N, Noggin; R, R-Spondin1; W, WNT3A; F, FGF4; 2D, initial monolayer culture; HI, human intestinal epithelium samples.)

Mentions: Both the CHIR and CHIR+F cells formed organoids also in the EN-condition, in the absence of RSPO1 (Fig 4) (S3 and S4 Figs). FGF4 inhibited the formation of organoids from the CHIR cells, whereas the addition of RSPO1 and WNT3A increased it (Fig 4C). There were no evident differences in the proportions of budding vs. bubble-like morphologies between the tested conditions (Fig 4D). Hematoxylin-eosin stainings revealed that the organoids consisted of both well-polarized epithelium and epithelium with weaker organization (Fig 4E). No differences were evident between test conditions. All organoid cultures matured to contain intestinal-like cell types, as evidenced by KRT20, CHRA, MUC2 and LYZ positive cells (Fig 4F and 4G) (S4 Fig). The cultures contained both E-CADHERIN positive epithelium and VIM positive mesenchyme (Fig 4F) (S4 Fig) and the organoids presented proliferative versus differentiated regions, as evidenced by KRT20/KI67 staining (Fig 4F) (S4 Fig). The intestinal differentiation markers CDX2, KRT20, KLF5 and IFABP2 were expressed at levels comparable to human intestinal epithelium in the EN, ENR and ENRW conditions, but weakly in the ENRF-condition (Fig 5A).


Intestinal Commitment and Maturation of Human Pluripotent Stem Cells Is Independent of Exogenous FGF4 and R-spondin1.

Tamminen K, Balboa D, Toivonen S, Pakarinen MP, Wiener Z, Alitalo K, Otonkoski T - PLoS ONE (2015)

HPSC-derived organoids express intestinal marker genes at levels comparable to human intestinal epithelium.Results represent day 33 organoids (d42 from hPSC) derived from day 9 CHIR cells. A. Gene expression of intestinal differentiation markers CDX2, KRT20, KLF5, IFABP2 and HOXA13. B. Expression of crypt area/intestinal stem cell markers LGR5, ASCL2 and SOX9. Data represent the mean ± SEM of 3–9 samples. (E, EGF; N, Noggin; R, R-Spondin1; W, WNT3A; F, FGF4; 2D, initial monolayer culture; HI, human intestinal epithelium samples.)
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4521699&req=5

pone.0134551.g005: HPSC-derived organoids express intestinal marker genes at levels comparable to human intestinal epithelium.Results represent day 33 organoids (d42 from hPSC) derived from day 9 CHIR cells. A. Gene expression of intestinal differentiation markers CDX2, KRT20, KLF5, IFABP2 and HOXA13. B. Expression of crypt area/intestinal stem cell markers LGR5, ASCL2 and SOX9. Data represent the mean ± SEM of 3–9 samples. (E, EGF; N, Noggin; R, R-Spondin1; W, WNT3A; F, FGF4; 2D, initial monolayer culture; HI, human intestinal epithelium samples.)
Mentions: Both the CHIR and CHIR+F cells formed organoids also in the EN-condition, in the absence of RSPO1 (Fig 4) (S3 and S4 Figs). FGF4 inhibited the formation of organoids from the CHIR cells, whereas the addition of RSPO1 and WNT3A increased it (Fig 4C). There were no evident differences in the proportions of budding vs. bubble-like morphologies between the tested conditions (Fig 4D). Hematoxylin-eosin stainings revealed that the organoids consisted of both well-polarized epithelium and epithelium with weaker organization (Fig 4E). No differences were evident between test conditions. All organoid cultures matured to contain intestinal-like cell types, as evidenced by KRT20, CHRA, MUC2 and LYZ positive cells (Fig 4F and 4G) (S4 Fig). The cultures contained both E-CADHERIN positive epithelium and VIM positive mesenchyme (Fig 4F) (S4 Fig) and the organoids presented proliferative versus differentiated regions, as evidenced by KRT20/KI67 staining (Fig 4F) (S4 Fig). The intestinal differentiation markers CDX2, KRT20, KLF5 and IFABP2 were expressed at levels comparable to human intestinal epithelium in the EN, ENR and ENRW conditions, but weakly in the ENRF-condition (Fig 5A).

Bottom Line: The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids.FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage.Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro.

View Article: PubMed Central - PubMed

Affiliation: Research Programs Unit, Molecular Neurology and Biomedicum Stem Cell Centre, University of Helsinki, Helsinki, Finland.

ABSTRACT
Wnt/beta-catenin signaling plays a central role in guiding the differentiation of the posterior parts of the primitive gut tube into intestinal structures in vivo and some studies suggest that FGF4 is another crucial factor for intestinal development. The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids. The most prominent induction of the well-established intestinal marker gene CDX2 was achieved when hPSC-derived definitive endoderm cells were treated with Wnt agonist molecule CHIR99021 during differentiation to hindgut. FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage. When hindgut stage cells were further cultured in 3D, they formed self-renewing organoid structures containing all major intestinal cell types even without exogenous R-spondin1 (RSPO1), a crucial factor for the culture of epithelial organoids derived from adult intestine. This may be explained by the presence of a mesenchymal compartment in the hPSC-derived organoids. Addition of WNT3A increased the expression of the Paneth cell marker Lysozyme in hPSC-derived organoid cultures, whereas FGF4 inhibited both the formation and maturation of intestinal-like organoids. Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro.

No MeSH data available.


Related in: MedlinePlus