Final Report Summary - MMSR (The Molecular Mechanisms of Stem Cell Self Renewal)
The Role Of Endogenous Wnt Signals In Self-Renewal And Differentiation Of Pluripotent Stem Cells
Derk ten Berge, Erasmus MC Stem Cell Institute, Dept of Cell Biology, Erasmus MC, Rotterdam, the Netherlands. d.tenberge@erasmusmc.nl
www.erasmusmc.nl/cellbiology/research/research-groups/berge/
Pluripotent stem cells can generate all cell types of the body and hold great potential for transplantation medicine, disease modelling, and the study of early development. Both the maintenance of the pluripotent state as well as differentiation towards specific tissue lineages are dependent on signalling cues from the environment. Protein growth factors constitute such signalling cues, and in this study we investigated the role of signalling proteins belonging to the Wnt family in the self-renewal and differentiation of both human and mouse pluripotent stem cells.
Pluripotent stem cells exist in naïve and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs). In the naïve state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naïve to primed pluripotency therefore represents a pivotal event in cellular differentiation. But what are the signals that control this fundamental differentiation step? Our research has demonstrated that Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that the source of these Wnt proteins is the ESCs themselves, explaining why their essential role had previously been overlooked. Furthermore, we have shown that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including from non-permissive mouse strains. Our results demonstrate that endogenous Wnt signals, produced by the ESCs themselves and acting in an auto- and paracrine manner, are essential and limiting ESC self-renewal factors and regulate the naïve-to-primed pluripotency transition.
The role for Wnt signals in mouse ESCs is not necessarily conserved in human ESCs since these differ in multiple aspects from mouse ESCs. In fact, human ESCs show many similarities to mouse EpiSCs, and are thought to possess a state of primed pluripotency. The hitherto unappreciated requirement for Wnt signals for mouse ESC self-renewal may therefore have implications for the establishment of naïve pluripotent cells from other species, including humans. This would be important for improving the reprogramming of adult human cells to the pluripotent state.
We next investigated the role of endogenous Wnt signals in self-renewal and directed differentiation of human ESCs and their mouse primed pluripotent counterpart, EpiSCs. A major obstacle towards therapeutic applications of human embryonic stem cells (hESCs) is limited control over their differentiation. In vivo, differentiation of the pluripotent epiblast occurs after implantation of the embryo during the process of gastrulation. Signalling proteins belonging to the Bmp and Wnt families are key gastrulation factors that mediate induction of the primitive streak in the embryo, and can induce primitive streak derivatives in human and mouse ESCs. However, spontaneous differentiation is prevalent, even in self-renewing conditions, and growth factor-induced differentiation generates mixtures of cell types. For example, Bmp4 induces trophoblast in addition to mesoderm, complicating efforts to obtain single desired lineages.
In this study we reveal endogenous Wnt proteins, produced by the hESCs and EpiSCs themselves, as hidden mediators of differentiation decisions. We identify Wnt signals as Bmp targets that induce the gastrulation factors Nodal, Fgf8 and Wnt3 itself and are required and sufficient for mesoderm induction. In contrast, induction of trophoblast differentiation by Bmp4 is Wnt independent, enabling the exclusive differentiation towards either individual lineage. In addition, we find that endogenous Wnt signals induce loss of pluripotency in hESCs and in their murine counterpart, epiblast stem cells (EpiSCs), and Wnt inhibition obviates the need to manually remove differentiated cells to maintain cultures. Moreover, Wnt-inhibited cultures differentiate more efficiently following directed differentiation. In EpiSCs, Wnt inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras.
Our findings show that endogenous Wnt signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs. A surprising twist in our findings is that Bmp4 induces both mesoderm as well as trophoblast-committed cells from hESCs, but only the induction of mesoderm-committed cells depends on the induction of Wnt genes by Bmp4. We further show that endogenous Wnt signals interfere with self-renewal of human ESCs and mouse EpiSCs. Endogenous Wnts push forward the aggregate developmental phenotype of EpiSCs to that reminiscent of late-gastrula stage epiblast, consisting of a mixture of genuine EpiSCs with cells in various stages of differentiation, including cells committed to the definitive endoderm lineage. Wnt inhibition prevents the induction of differentiation genes and commitment to the definitive endoderm fate, thereby maintaining a high percentage of genuine EpiSCs displaying their pre-gastrula phenotype, as evidenced by their contribution to blastocyst chimeras. A similar process takes place in hESCs, where we show that Wnt inhibition is so effective in suppressing differentiation that it obviates the need for manual removal of differentiated cells. This greatly reduces the efforts and skills required to work with hESCs.
Our findings have obvious ramifications for the guided differentiation of hESCs. For instance, to induce trophoblast one should stimulate with Bmp4 in the presence of a Wnt inhibitor to avoid induction of mesoderm. Conversely, mesoderm is best obtained using Wnt3a in lieu of Bmp4 to avoid trophoblast induction. Furthermore, Wnt-inhibited hESCs differentiate more efficiently to the trophoblast lineage, indicating that genuine EpiSCs and hESCs, maintained as homogeneous undifferentiated populations by Wnt inhibition, are superior substrates for differentiation as they contribute fewer undesired lineages to the population. We also find that different cell lines and culture media display various tendencies for endogenous Wnt-induced differentiation, affecting their suitability for specific purposes such as mesoderm or neural differentiation. Lastly, ESCs are commonly aggregated into embryoid bodies for the derivation of mesendodermal lineages. We now demonstrate that embryoid bodies mediate the transition of mouse ESCs into EpiSCs, followed by activation of an endogenous Wnt gradient and mesendodermal induction. A more controlled way of inducing mesendodermal lineages would be by directly inducing genuine EpiSCs with defined levels of Wnt signals.
Derk ten Berge, Erasmus MC Stem Cell Institute, Dept of Cell Biology, Erasmus MC, Rotterdam, the Netherlands. d.tenberge@erasmusmc.nl
www.erasmusmc.nl/cellbiology/research/research-groups/berge/
Pluripotent stem cells can generate all cell types of the body and hold great potential for transplantation medicine, disease modelling, and the study of early development. Both the maintenance of the pluripotent state as well as differentiation towards specific tissue lineages are dependent on signalling cues from the environment. Protein growth factors constitute such signalling cues, and in this study we investigated the role of signalling proteins belonging to the Wnt family in the self-renewal and differentiation of both human and mouse pluripotent stem cells.
Pluripotent stem cells exist in naïve and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs). In the naïve state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naïve to primed pluripotency therefore represents a pivotal event in cellular differentiation. But what are the signals that control this fundamental differentiation step? Our research has demonstrated that Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that the source of these Wnt proteins is the ESCs themselves, explaining why their essential role had previously been overlooked. Furthermore, we have shown that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including from non-permissive mouse strains. Our results demonstrate that endogenous Wnt signals, produced by the ESCs themselves and acting in an auto- and paracrine manner, are essential and limiting ESC self-renewal factors and regulate the naïve-to-primed pluripotency transition.
The role for Wnt signals in mouse ESCs is not necessarily conserved in human ESCs since these differ in multiple aspects from mouse ESCs. In fact, human ESCs show many similarities to mouse EpiSCs, and are thought to possess a state of primed pluripotency. The hitherto unappreciated requirement for Wnt signals for mouse ESC self-renewal may therefore have implications for the establishment of naïve pluripotent cells from other species, including humans. This would be important for improving the reprogramming of adult human cells to the pluripotent state.
We next investigated the role of endogenous Wnt signals in self-renewal and directed differentiation of human ESCs and their mouse primed pluripotent counterpart, EpiSCs. A major obstacle towards therapeutic applications of human embryonic stem cells (hESCs) is limited control over their differentiation. In vivo, differentiation of the pluripotent epiblast occurs after implantation of the embryo during the process of gastrulation. Signalling proteins belonging to the Bmp and Wnt families are key gastrulation factors that mediate induction of the primitive streak in the embryo, and can induce primitive streak derivatives in human and mouse ESCs. However, spontaneous differentiation is prevalent, even in self-renewing conditions, and growth factor-induced differentiation generates mixtures of cell types. For example, Bmp4 induces trophoblast in addition to mesoderm, complicating efforts to obtain single desired lineages.
In this study we reveal endogenous Wnt proteins, produced by the hESCs and EpiSCs themselves, as hidden mediators of differentiation decisions. We identify Wnt signals as Bmp targets that induce the gastrulation factors Nodal, Fgf8 and Wnt3 itself and are required and sufficient for mesoderm induction. In contrast, induction of trophoblast differentiation by Bmp4 is Wnt independent, enabling the exclusive differentiation towards either individual lineage. In addition, we find that endogenous Wnt signals induce loss of pluripotency in hESCs and in their murine counterpart, epiblast stem cells (EpiSCs), and Wnt inhibition obviates the need to manually remove differentiated cells to maintain cultures. Moreover, Wnt-inhibited cultures differentiate more efficiently following directed differentiation. In EpiSCs, Wnt inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras.
Our findings show that endogenous Wnt signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs. A surprising twist in our findings is that Bmp4 induces both mesoderm as well as trophoblast-committed cells from hESCs, but only the induction of mesoderm-committed cells depends on the induction of Wnt genes by Bmp4. We further show that endogenous Wnt signals interfere with self-renewal of human ESCs and mouse EpiSCs. Endogenous Wnts push forward the aggregate developmental phenotype of EpiSCs to that reminiscent of late-gastrula stage epiblast, consisting of a mixture of genuine EpiSCs with cells in various stages of differentiation, including cells committed to the definitive endoderm lineage. Wnt inhibition prevents the induction of differentiation genes and commitment to the definitive endoderm fate, thereby maintaining a high percentage of genuine EpiSCs displaying their pre-gastrula phenotype, as evidenced by their contribution to blastocyst chimeras. A similar process takes place in hESCs, where we show that Wnt inhibition is so effective in suppressing differentiation that it obviates the need for manual removal of differentiated cells. This greatly reduces the efforts and skills required to work with hESCs.
Our findings have obvious ramifications for the guided differentiation of hESCs. For instance, to induce trophoblast one should stimulate with Bmp4 in the presence of a Wnt inhibitor to avoid induction of mesoderm. Conversely, mesoderm is best obtained using Wnt3a in lieu of Bmp4 to avoid trophoblast induction. Furthermore, Wnt-inhibited hESCs differentiate more efficiently to the trophoblast lineage, indicating that genuine EpiSCs and hESCs, maintained as homogeneous undifferentiated populations by Wnt inhibition, are superior substrates for differentiation as they contribute fewer undesired lineages to the population. We also find that different cell lines and culture media display various tendencies for endogenous Wnt-induced differentiation, affecting their suitability for specific purposes such as mesoderm or neural differentiation. Lastly, ESCs are commonly aggregated into embryoid bodies for the derivation of mesendodermal lineages. We now demonstrate that embryoid bodies mediate the transition of mouse ESCs into EpiSCs, followed by activation of an endogenous Wnt gradient and mesendodermal induction. A more controlled way of inducing mesendodermal lineages would be by directly inducing genuine EpiSCs with defined levels of Wnt signals.