Capturing Cardiogenesis in Gastruloids

Organoids are powerful models for studying tissue development, physiology, and disease. However, current culture systems disrupt the inductive tissue-tissue interactions needed for the complex morphogenetic processes of native organogenesis. Here, we show that mouse embryonic stem cells (mESCs) can be coaxed to robustly undergo fundamental steps of early heart organogenesis with an in-vivo-like spatiotemporal fidelity. These axially patterned embryonic organoids (gastruloids) mimic embryonic development and support the generation of cardiovascular progenitors, including first and second heart fields. The cardiac progenitors self-organize into an anterior domain reminiscent of a cardiac crescent before forming a beating cardiac tissue near a putative primitive gut-like tube, from which it is separated by an endocardial-like layer. These findings unveil the surprising morphogenetic potential of mESCs to execute key aspects of organogenesis through the coordinated development of multiple tissues. This platform could be an excellent tool for studying heart development in unprecedented detail and throughput.

Definition of in Vitro Microenvironments to Characterize and Control Pancreatic Progenitor Expansion and Differentiation

Chiara Greggio

The pancreas plays a central role in metabolism. The exocrine pancreas, composed of ductal and acinar cells, secretes and delivers digestive enzymes into the duodenum where they contribute to food digestion. The endocrine pancreas, constituted by the islets of Langerhans, secretes hormones in the blood to control glucose levels. In particular, insulin is the hormone produced by β-cells that instructs the peripheral tissues to uptake circulating glucose. Diabetes mellitus is a heterogeneous disease characterized by deregulated glucose homeostasis due to an impaired insulin function. The advancements in clinical practice have made of diabetes a chronic, instead of a lethal, disease; yet no definitive cure is available. Beta-cell transplantation offers promising results, especially for type 1 diabetes where β-cells are selectively eliminated by an autoimmune attack. Unfortunately its application suffers from the scarcity of transplantable pancreas/islets from cadaveric donors. Human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) would constitute unlimited sources of transplantable β-cells, but the available differentiation protocols are not yet optimal, especially with regards to the numbers and the functionality of the generated β-cells. It is anticipated that a stepwise protocol would succeed in generating mature β-cells in vitro if it is capable of fully mimicking the embryonic development of these cells. Many aspects of pancreatic organogenesis have been elucidated and could serve as a guide for driving the commitment to β-cells. Unfortunately, pancreatic progenitors in vitro do not behave as in the intricate context of an embryo. This severely limits both the understanding of pancreatic development at the single-cell level and our ability in manipulating the process. The aim of our work was to develop in vitro methodologies to sustain pancreatic progenitor culture and expansion and to establish conditions to manipulate their progeny. To this aim, we combined an informed approach based on developmental biology and an empirical one. We first observed that pancreatic epithelial explants from embryonic day 10.5 mice could be cultured in presence of Matrigel™ and exogenous FGFs even in the absence of the mesenchyme that normally surrounds the epithelium. Notably, the removal of mesenchyme did not affect the endocrine commitment pattern of pancreatic progenitors. We then defined culture conditions allowing for the expansion of dissociated embryonic pancreatic progenitors in a three-dimensional Matrigel™-based environment. Under these conditions, progenitors proliferated and self-organized to generate pancreatic organoids containing both progenitors and differentiated cells, mostly exocrine, after 7 days of culture. When grafted into recipient pancreatic explants, the cultured progenitors contributed to the three epithelial pancreatic lineages (ductal, endocrine, acinar), integrating seamlessly into the endogenous cellular network. Thus, cultured progenitors retained their potential to differentiate into endocrine cells, but expressed it only in the appropriate niche. Subsequent removal of single components from the culture system led to the identification of some essential requirements for the maintenance/expansion of dissociated pancreatic progenitors, such as a strong FGF signaling and the Rho-associated kinase (ROCK) inhibitor Y-27632. In addition to this, we observed that only small clusters of pancreatic progenitors, but not single cells, were able to generate pancreatic organoids, suggesting a pivotal role for intercellular signaling in progenitor maintenance and expansion. By manipulating the components of the medium, we could affect fate commitment. In particular, the removal of FGF1 increased the yield of endocrine cells generated in vitro. In our search for a full control over the in vitro niche, we explored chemically defined matrices to replace Matrigel™. We showed that pancreatic progenitors could be cultured on laminin-functionalized hydrogels, although less efficiently than in Matrigel™. Moreover, differentiation into exocrine and endocrine cells occurred spontaneously under these conditions. By comparing Matrigel™, hydrogel microwells and two-dimensional culture systems, we uncovered the importance of a tridimensional architecture for pancreatic progenitor maintenance. We showed that pancreatic progenitors lost their identity as they flattened onto 2D surfaces, whereas 3D culture systems maintained the epithelial character of pancreatic progenitors and allowed the acquisition of apical polarization. To conclude, our work provides the first detailed characterization of in vitro culture systems for expansion and manipulation of dissociated embryonic pancreatic progenitors. Further implementation will hopefully allow the establishment of a fully-defined in vitro niche for developing pancreas with potential fundamental implications for the expansion of ES-derived pancreatic progenitors and their differentiation into functional β-cells.

EPFL2011

In vitro modeling of complex microenvironmental regulation of stem cells

Yoji Tabata

Although stem cells hold tremendous potential for clinical applications, their in vitro manipulation remains very challenging. In vivo, stem cells reside in intricate 3D microenvironments, termed niche, in which many local and systemic extrinsic factors are integrated by the cells to induce controlled fate choices. Niche factors must not only be presented to stem cells in the correct composition, but also in a dynamic and spatially heterogeneous manner to evoke the appropriate response. However, state-of-the-art in vitro culture platforms fail to capture such microenvironmental complexity. In this thesis, a novel hydrogel-based microchip technology was invented to recapitulate in vitro the complexity of native stem cell niches. In this system, stem cells can be exposed to gradients of soluble or extracellular matrix (ECM)-tethered biomolecules. As a first proof of principle, this platform was used to locally influence the behavior of mouse embryonic stem cells (mESC) by exposing them to soluble, matrix-tethered or cell-secreted leukemia inhibitory factor (LIF). mESCs grown in synthetic 3D gels were found to respond to LIF gradients in a spatially regulated manner. In two subsequent studies, the microchip technology was utilized to recapitulate embryonic processes that are characterized by highly dynamic and spatially controlled morphogenetic processes that can currently not be modelled in a 3D context in vitro. First, an in vitro model of the developing central nervous system was conceived. In the developing vertebrate neural tube, dorsoventral identity of neural progenitors is defined by counter gradients of sonic hedgehog (Shh) and bone morphogenetic protein 7 (BMP7). Using the developing neural plate of chick embryos as a model system, a dedicated microchip was developed for exposing the neural progenitors in these tissues to gradients of Shh and BMP7. This approach allowed achieving, for the first time, ex vivo dorsoventral patterning. Secondly, a similar approach was employed towards modeling early human development in vitro. To this end, colonies of human embryonic stem cells (hESC) were exposed to opposing gradients of BMP4 and Noggin which induced mesendoderm differentiation exclusively in specific regions of the multicellular constructs. This study revealed the power of the developed platform to spatially control hESCs self-patterning. In a last set of experiments, the microchip technology was applied to mimic the complex molecular and cellular make-up of the bone marrow niche that regulates the activity of hematopoietic stem cells (HSC). A microchip system was successfully designed to capture the main anatomic features of native bone marrow niches with spatially separated compartments that supported either HSC dormancy or activation. Taken together, this thesis presents a set of innovative microengineering concepts for recapitulating the spatiotemporal complexity of native stem cell microenvironments. The platforms introduced here represent important additions to the currently rather limited set of in vitro tools for the study of complex multicellular phenomena in developmental biology, tissue engineering and regenerative medicine.

EPFL2017

KRAB/KAP1 Regulation of Embryonic Stem Cell Pluripotent Self-renewal

Andrea Corsinotti

KRAB-ZFPs constitute the largest family of transcription factors (TFs) encoded by the mouse genome and are known to interact with the co-repressor KAP1. KRAB/KAP1-mediated regulation is essential for several development- and ESC- specific functions, in particular regulation of pluripotent self-renewal. We performed an expression analysis in mouse pluripotent ESCs and differentiated cells. A large number of KRAB-ZFPs was expressed in pluripotent cells and many of them were differentially regulated in other cell types, suggesting that a set of KRAB-ZFPs can be involved in ESC-specific functions. We identified a set of candidate genes, including Zfp459, which show a pluripotency-specific expression pattern. Zfp459 depletion in ESCs induced transcriptional perturbation of pluripotency- and differentiation-associated genes, even if their ability to self-renew or to differentiate was not impaired, and its expression was not required for the formation of blastocysts ex vivo. To understand the precise role of Zfp459 in ESCs further experiments for the identification of its targets are still required. We used a lentiviral vector (LV) to over-express in ESCs ZFP57, which had been previously demonstrated to play a role in regulating genomic imprinting, in fusion with HA-tags. ChIP-seq experiments using a HA antibody demonstrated that ZFP57-HA binds all the known and predicted imprinted loci and identified a hexanucleotide consensus in most of the ZFP57 binding sites co-occupied also by KAP1 and its associated factor SetDB1. We also demonstrated that our LV based expression system could be exploited to perform ChIP-seq analyses in ESCs of TFs, such as Zfp459, for which specific antibodies are not available. Finally, when we cultured ESCs in different conditions, we observed that KAP1 recruitment upstream of Nanog, which had been previously observed, was dependent on the MAPK and GSK3 signaling pathways. We hypothesized that this could be due to the presence in these conditions of ESCs expressing heterogeneous Nanog levels. However, when Nanoghigh and Nanoglow cells were sorted, both the populations shown a comparable enrichment for KAP1 binding upstream of Nanog. The precise role for the KAP1-mediated regulation of Nanog in ESCs and its dependence on extrinsic signaling pathways remains unclear, even if we hypothesize that it can depend on the mono-/bi-allelic expression of Nanog in ESCs and during early development.

EPFL2012