In vitro modeling of complex microenvironmental regulation of stem cells

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.

Bioengineering approaches to emulate the stem cell niche

Sylke Höhnel

Stem cell therapies hold tremendous potential for tissue and organ regeneration. Yet, there remains significant need for better ex vivo culture and manipulation methods. On the one hand, many tissue-specific stem cells cannot be propagated without causing rapid deviations in cellular phenotype. On the other hand, outside of a developing organism, it remains very difficult to differentiate stem cells into mature functional cell types. Such protocols could help to build more effective and predictive pre-clinical models that would reduce or even replace animal studies. Manipulating a stem cell in vitro requires a thorough understanding of its microenvironment, or niche, and how niche factors influence stem cell fate. A customizable microarray platform was therefore developed to examine the interplay of biochemical and mechanical signals on stem cell behavior. This array was implemented to study combinations of 12 different proteins with varying substrate stiffness and their effect on the adipogenic differentiation of human mesenchymal stem cells (MSCs). These cells and their differentiated progenies are of high therapeutic relevance for skeletal tissue regeneration and have also been implied as niche-modulating neighbors of hematopoietic stem cells (HSCs) in the bone marrow (BM). Using this combinatorial approach, substrate stiffness was found to be the major determinant of the frequency of lipid accumulation in MSCs, while the lipid content was found to be strongly driven by the biochemical context. To further understand how niche cells together with stem cells self-organize to form a functional tissue, three-dimensional (3D) organ-mimicking structures from spheroids of pluri- and multi-potent have recently been developed. These systems, termed organoids, are expected to provide instrumental insights into tissue or organ development and they could be of great use for disease-specific drug screenings and to generate clinically transplantable tissues. However, current protocols for organoid generation remain highly inefficient and irreproducible. To perform the high-throughput aggregation of one or multiple cell types and to allow their long-term culture, a high-density array of U-shaped microwells was developed. This platform was used to initiate 3D co-cultures of the hematopoiesis-supportive cell line OP9 together with HSCs. To expand on the potential of this co-culture model to study more relevant primary BM cells, uniform clonal mesensphere cultures from human MSCs were established. However, restricted knowledge about the phenotypic identity of both niche and stem cell is a major limitation to initiate these co-cultures in vitro. Therefore, in this thesis a cell-targeting approach through covalent SNAP-tag-based cell-pairing is proposed. Specifically, SNAP-tag fusion proteins expressed on cell membranes were targeted to form covalent bonds with substrates that harbor the SNAP-tag substrate benzylguanine (BG). Immobilizing sufficient amounts of BG on cellular membranes is a mean to enable cell-cell pairing as demonstrated by the covalent aggregation of microbeads carrying SNAP-tag and BG at high concentrations. The artificial niche models developed in this study are broadly applicable to desired cellular systems. These approaches help to gain insight into the multifactorial composition and architectural organization of specific niches and to enable their reconstruction in vitro.

EPFL2015

Modeling hematopoietic stem cell dynamics in bioengineered niches

Sonja Giger

Bone marrow transplantation is a well-established medical procedure for the treatment of various hematologic diseases. However, the relatively low number of hematopoietic stem cells (HSCs) that can be harvested, especially from umbilical cord blood, limits even broader applicability of the procedure. A better understanding of the biology of HSCs, and in particular how these rare cells are regulated by microenvironmental niches in the bone marrow, could ultimately enable robust in vitro expansion of HSCs. In this thesis, several novel strategies were developed to study and manipulate HSC biology in vitro, through the regulation of key niche factors, cellular metabolism, and the complex multicellular crosstalk in a niche-mimicking context. First, we made use of gastruloids, an embryonic stem cell-based model of early mouse development, to mimic key aspects of embryonic hematopoiesis in vitro. Simultaneously with the establishment of a vascular network, we detected by immunophenotyping the emergence of primitive blood progenitor cells during the late developmental stages of gastruloids. These embryonic blood progenitors were spatially localized close to a vascular-like plexus in the anterior portion of the gastruloid. Colony-forming assays demonstrated an interesting differentiation potential of these cells. These data demonstrate the potential of gastruloids as an easily accessible in vitro model to study blood development in an embryo-like context. Second, a bioengineering approach was used to study HSC fate choices upon exposure to niche-specific ligands in a well-defined artificial environment. A combination of time-lapse microscopy and single-cell multigene expression analysis was used to define differentiation and cell-cycle states of mouse and human HSCs. Strikingly, selected artificial niches reduced proliferation and maintained the long-term multilineage potential of HSCs in vitro. These artificial niches hold significant potential for the study of mechanisms involved in HSC fate regulation. Third, the influence of the metabolism on fate choices of adult HSCs was studied, specifically focusing on fatty acid Î²-oxidation. Malonyl-CoA was used to reversibly block fatty acid Î²-oxidation in mouse HSCs. In vitro treatment of HSCs with malonyl-CoA promoted their expansion and increased lymphoid reconstitution upon in vivo transplantation. This study sheds new light on the role of the metabolic environment in HSC regulation. Surprisingly, exposure to only a single, readily available metabolite was found to be sufficient to strongly influence HSC behavior. Fourth, a miniaturized multicellular culture system was developed to mimic the hallmarks of the dynamics of HSCs in their native bone marrow. Primary human mesenchymal stem/progenitor cells and endothelial cells were aggregated in a high-throughput manner in biomimetic hydrogel microwells to form self-organizing bone marrow organoids. Immunostaining demonstrated the formation of branched vascular networks, rendering the organoids permissive for the homing of human hematopoietic stem and progenitor cells. These results suggest that bone marrow organoids may be a suitable platform for the modeling of physiological and clinically-relevant stem cell dynamics in vitro. Altogether, this thesis presents several innovative approaches for modeling key features of native stem cell microenvironments that will contribute to a better understanding of the biology of HSCs and their regulatory niche.

EPFL2019

Photopatterning of Synthetic Hydrogels

Katarzyna Mosiewicz

In recent years sophisticated biomaterials have made a significant impact on our understanding of cellular behavior, particularly in the fields of stem cell research and tissue engineering. Nevertheless, the promising potential of stem cells for regenerative medicine has remained largely unmet. One reason for such limitations could be that there is still incomplete understanding of stem cell behavior due to a lack of advanced in vitro tools suitable to achieve adequate control over cellular processes. By mimicking the dynamic and complex biophysical and biochemical interactions between cells and their natural extracellular matrixes (ECMs), it is possible that we may bridge this gap between current limited in vitro models and complex in vivo organisms. In this thesis, an innovative class of biomaterials is presented that enables the flexible in vitro recapitulation of the dynamic biochemical and biophysical signaling involved in controlling cell fate. Such systems of increasing fidelity to real physiological processes could bring new insights into fundamental cell biology as well as bring us to closer towards cell-based therapeutics applications. In order to achieve a dynamic artificial ECM (aECM), we designed chemical schemes which would allow us to control the biochemical and biophysical properties of the hydrogel in space, time and intensity. The chosen concept is built on a synthetic hydrogel providing static physicochemical support, while spatiotemporal control over a single or multiple signals is achieved by spatiotemporal photoactivation reactions within this hydrogel. Indeed, by modulating the duration, location and intensity of illumination, desired heterogeneities in biochemical and biophysical signal can be introduced in the otherwise homogeneous and static hydrogel network. To implement the designed hydrogel photo-patterning tool, new chemical components were synthesized. Synthetic hydrogels based on poly (ethylene glycol) (PEG) were utilized due to their inert properties, cell biocompatibility and lack of unspecific protein binding. Biochemical signal tethering was rendered spatiotemporally controllable by adding photosensitivity to the existing enzyme-based crosslinking scheme. This was achieved by functionalizing one of enzymatic substrate with a photo-labile “caging” moiety, thereby preventing crosslinking to occur until light illumination. This caged substrate was incorporated into the hydrogel network and served as a light-controllable and specific ligand-binding site. Importantly, it was possible to photo-pattern proteins, which are much complex than peptide-based biomolecules, and their bioactivity was fully preserved with this site-specific immobilization scheme. It was also possible to pattern biophysical cues by making the Michael-type (MT) addition reaction for crosslinking PEG-based hydrogel photosensitive. In this case, the thiol moieties of one of the reactive PEG macromers undergoing crosslinking, were equipped with a caging group which prevented its susceptibility to the counter-reactive functionality, the vinyl sulfone group. Thus, the crosslinking density of the hydrogel backbone could be controlled by light, which was directly translated into differential patterns of hydrogel stiffness. Using this approach, user-defined biochemical and biophysical patterns and even gradients could be obtained in the spatiotemporal manner within the same hydrogel. The crosslinking reaction is the critical parameter in the success of hydrogel photo-patterning, and must rely on a well-controlled and highly selective chemical scheme. Therefore, we also developed a novel enzyme-based cross-linking scheme for synthetic PEG hydrogels. The reaction originates from the naturally occurring post-translational modification of proteins by phosphopantetheine (Ppant) group performed by a family of enzymes known as Ppant transferases (PPTases). By synthesizing biochemical components of this reaction, we implemented a PPTase-catalyzed reaction to form and bio-functionalize PEG hydrogel. The utility of the developed hydrogel platforms was determined in the context of 2D and 3D cell culture. Photo-directed patterns of a cell-adhesive fibronectin fragment triggered adhesion and spreading of muscle progenitor cells, a phenomenon which occurred only within the areas of patterned signaling protein. A photo-patterned elasticity gradient was used to direct mesenchymal stem cell spreading and migration. Finally, the PPTase-based hydrogel was proven to be suitable for 3D culture, as shown by encapsulation of primary human fibroblasts. In this thesis, we demonstrated that a combination of chemical schemes could be assembled to achieve a functional materials toolbox which would begin to reconstruct the complexity of the dynamic in vivo ECM. Such a tool is expected to contribute to the field of cell biology by allowing us to study questions which previously could not be addressed in vitro, and could ultimately contribute to cell-based regenerative medicine.

EPFL2012