Differential impact of TGF beta/SMAD signaling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGF beta pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGF beta family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGF beta pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGF beta/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGF beta/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.

FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo

Anne Grapin-Botton

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

2006

Evolution of the mechanisms and molecular control of endoderm formation

Daniel Constam, Anne Grapin-Botton

Endoderm differentiation and movements are of fundamental importance not only for subsequent morphogenesis of the digestive tract but also to enable normal patterning and differentiation of mesoderm- and ectoderm-derived organs. This review defines the tissues that have been called endoderm in different species, their cellular origin and their movements. We take a comparative approach to ask how signaling pathways leading to embryonic and extraembryonic endoderm differentiation have emerged in different organisms, how they became integrated and point to specific gaps in our knowledge that would be worth filling. Lastly, we address whether the gastrulation movements that lead to endoderm internalization are coupled with its differentiation.

Elsevier2007

Bioengineering the Human Muscle Stem-Cell Niche for Therapeutic Applications

Omid Mashinchian

In spite of decades of research, no feasible method for obtaining sufficient numbers of uncommitted muscle stem cells (MuSCs) for therapy of degenerative muscle diseases exists. One of the most fundamental problems associated with stem cell therapy of muscle is that removal of MuSCs from their tissue microenvironment and expansion in conventional culture induces their terminal commitment to myogenic differentiation. This in-vitro loss of stemness impairs the long-term engraftment potential of MuSCs and renders them unsuitable for stem cell therapy. In another disease relevant context, aging, dramatic changes in the functionality of MuSCs have been shown to depend on an altered composition of their microenvironment. Thus, muscle progenitors are fundamentally dependent on their local environment, commonly known as the âstem cell niche", and a better understanding of its role in guiding stem cell function is highly relevant for the development of therapies for aging and muscle diseases. The muscle stem cell niche is composed of diverse cellular and acellular elements, including different supportive cell types, growth factors and extracellular matrix, and as outlined above, is critical for maintaining healthy stem cell characteristics such as the capacity for self-renewal, commitment, and differentiation. Using molecular biology approaches, we decided to interrogate (I) the role of the cellular environment on stem cell commitment, (II) to test whether, in a physiological context, the systemic circulation has niche-independent effects on MuSC stemness, and (III) to study a population of MuSC-supportive cells that is affected by the aging process. In the first study of this thesis, we have successfully developed an organoid-like-approach for the scalable derivation of uncommitted MuSCs from human induced pluripotent stem cells (iPSCs) in a biologically faithful cellular 3D-environment in suspension embryoids. To this end, we employed human iPSCs and a spectrum of immortalized cell lines to screen for 3D-aggregation conditions promoting mesoderm formation and subsequent specification to the myogenic lineage without the parallel upregulation of myogenic commitment markers. Compared to myogenic cells derived from adult human skeletal muscle, this niche-mimetic embryoid derived progenitors display significantly enhanced engraftment into the muscle stem cell position peripheral to muscle fibers, and restoration of dystrophin expression when transplanted into muscles of a mouse model of Duchenne muscular dystrophy. In a second study, we present a novel method for encapsulation of human muscle progenitors in highly diffusible polyethersulfone hollow fiber capsules to identify highly specific "transcriptional-signature" induced by systemic aging in-vivo. This technique allows to study the systemic circulation in health, disease and aging at an unprecedented level in human cell types of choice. Finally, in our third study, we investigated the cellular cross-talk between muscle stem cells and non-myogenic niche-based cell type, called fibro/adipogenic progenitors (FAPs). Interestingly, the support-function and the cross-talk with stem cells are dramatically impaired in aged FAPs. We demonstrate that this mechanism can be targeted to rejuvenate myogenesis. Taken together, "mimicking" the physicochemical-interactions of MuSCs with their niche-resident cell types, represents a powerful opportunity to manipulate MuSC function in aging and disease.

EPFL2019

Bioengineering the Human Muscle Stem-Cell Niche for Therapeutic Applications

Omid Mashinchian

EPFL2019