Elucidating the Role of Matrix Stiffness in 3D Cell Migration and Remodeling

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

Bioengineering stem cell niches in 2D and 3D

Nora Leonardi

Muscle degenerative disorders, such as Duchenne muscular dystrophy, have a profound impact on the quality of life and survival of patients. Transplantation of myoblasts to restore muscle function has been discouraging since these cells die and don't migrate but it has more recently been reported that muscle stem cells, known as satellite cells, have an extraordinary potential to contribute to muscle fiber regeneration, home to their niche and replenish the stem cell pool when injected freshly into damaged muscle. These cells however quickly lose their regenerative potential when cultured in vitro. Adult stem cells are known to be regulated by their microenvironment through a complex mixture of signals including soluble factors, matrix bound proteins, neural input and mechanical properties and the identification of extrinsic factors that can regulate muscle stem cell quiescence and self-renewal has been a focus of research in the Blau lab. Strikingly, recent studies in the Blau lab have shown that muscle stem cells cultured on compliant hydrogels coated with laminin were more viable than those cultured on tissue culture plastic and robustly contributed to muscle regeneration when transplanted into irradiated mouse leg muscles. The aim of this project was to follow up on these findings and to further elucidate the role biophysical and biochemical microenvironmental cues play in the regulation of muscle stem cell fate. Hydrogels have emerged as a popular material to recreate the stem cell niche in vitro due to their biocompatibility, high water content, tissue-like elasticity and diffusive properties and were employed here to fabricate substrates of varying rigidities. The physicochemical properties and tethered protein densities of a range of poyl(ethylene glycol) hydrogels were first characterized to define a suitable set of cell culture substrates. Gel stiffness could be linearly modulated from 2.5 to 45 kPa and the ligand density was found to be in the low femtomolar range. ELISA showed similar protein density on gels of different stiffnesses but immunofluorescence could not fully confirm this. Committed myoblasts were then seeded on these gels and shown to respond to substrate stiffness and ligand density in terms of morphology. Lastly, freshly isolated muscle stem cells were cultured on these gels and on rigid tissue culture plastic and shown to spread more, be more viable and grow more rapidly on stiffer gels as compared to soft ones. Myogenic progression was retrospectively analysed using immunohistochemistry but no differences between culture conditions were observed. Based on the influences of gel rigidity and ligand density observed in vitro, an in vivo assay, the ultimate test of stem cell function, is currently underway.

2009

In vitro Fate Mapping of Single Hematopoietic Stem Cells

Aline Roch

The in vitro expansion of hematopoietic stem cells (HSC) for clinical applications is hampered by a rapid loss of HSC blood reconstitution capability in culture. While these rare cells can be stimulated to massively proliferate, cell divisions mostly result in differentiation caused by the lack of interactions with the native microenvironment, termed niche, in the bone marrow. Indeed, an increasing body of literature highlights the crucial role of instructive niche signals directing HSC fate in vivo. But how HSC fate, and in particular the delicate choice between self-renewal and differentiation divisions, is controlled by niche signaling cues remains unknown. Therefore, the goal of this thesis was to systematically characterize HSC fate decisions in vitro, and to utilize this knowledge to design artificial niches that maintain stemness. Differentiating HSCs first give rise to several transient populations of highly proliferative multipotent progenitors. Because reliable markers that distinguish HSCs from the earliest multipotent progenitors in vitro are lacking, it is currently not possible to discriminate between HSC self-renewal and commitment fate choices. The development and application of novel tools to probe HSC fate at the single cell level is therefore crucial, even more so because HSC populations are highly heterogeneous. In the first part of this thesis, a micro-engineered single cell analysis platform was employed to track in high-throughput the fate of individual HSCs by time-lapse microscopy. Single cell imaging revealed a surprising direct generation of megakaryocytes, cells that produce blood platelets, from phenotypic HSCs in the complete absence of a cell division. Megakaryocytic differentiation has previously been shown to occur very early in hematopoiesis and recent studies revealed the existence of a subpopulation of HSCs that is predetermined to undergo megakaryocytic differentiation. Our results suggest that some megakaryocyte progenitors may not be directly derived from HSCs but rather share the same cell surface marker repertoire such that they are indistinguishable from HSCs by state-of-the-art purification strategies. In order to discriminate between HSC self-renewal and commitment at single cell level, in the second part of this thesis gene expression signatures associated with the stem cell and multipotent progenitor cell states were established. Twelve differentially expressed genes marking the quiescent HSC state were identified, including four genes encoding cellcell interaction signals in the niche. Single cell multigene expression analysis performed on daughter cells, derived from single HSCs in serum-free culture, showed a rapid loss of the HSC identity with increasing number of cell divisions. In order to prevent such a dramatic loss of stemness in vitro, biomimetic microenvironments were engineered to display ligands of the newly identified niche components. Exposure of single HSCs to these artificial niches revealed a reduction in the mitotic activity of HSCs. Strikingly, in vivo transplantation of artificial niche-cultured HSCs resulted in long-term blood reconstitution of irradiated mice, demonstrating maintenance of functional HSC in vitro when exposed to critical cell-cell interaction signals found in native niches. Studies with invertebrates have shown that the pool of stem cells in vivo is controlled through asymmetric self-renewal divisions, resulting in two daughter cells with distinct fates, only one of which maintains stemness. Very little is known about asymmetric divisions of HSCs. Therefore, in the third part of this thesis, the symmetry of HSC divisions was systematically investigated in vitro. Using time-lapse imaging and single cell multigene expression analysis of paired daughter cells isolated by micromanipulation, approximately one third of all HSC was found to divide in an asymmetric fashion under serum-free culture conditions. Strikingly, paired daughter cells of cultured HSCs that were activated in vivo to exit their dormant state were found to divide mostly in an asymmetric fashion. These results shed light on the critical role of the in vivo microenvironment in specifying HSC fate and in particular asymmetric cell divisions. Altogether, this thesis demonstrates the power of single cell analyses to unravel stem cell fate decisions, and presents a novel approach to identify functional artificial niches to maintain HSCs in culture. This experimental paradigm should contribute to the development of better strategies to study and manipulate other stem cell types in culture. One exciting avenue is the expansion of human cord blood-derived HSCs, a cellular source that is particularly plagued by a lack of sufficient numbers for transplantation, with the aim of improving HSC therapies.

EPFL2014

Microscale patterning of hydrogel stiffness through light-triggered uncaging of thiols

Laura Kolb, Matthias Lütolf, Katarzyna Mosiewicz

Mammalian cell behavior is strongly influenced by physical and chemical cues originating from the extracellular matrix (ECM). In vivo, ECM signals are displayed in a spatiotemporally complex fashion, often composed as gradients and in concentration profiles that change in time. Most in vitro models to study the role of ECM signals in regulating cell behavior are limited in capturing this microenvironmental complexity, as they are static and homogeneous. In order to achieve a dynamic control of the physical properties of a hydrogel network, we here designed a chemical scheme to control poly(ethylene glycol) (PEG) hydrogel stiffness in space, time and intensity. Specifically, we combined caging chemistry and Michael-type addition to enable the light-triggered local control of hydrogel crosslinking density. Thiol moieties of one of the reactive PEG macromers undergoing crosslinking were equipped with caging groups to prevent their susceptibility to the counter-reactive vinyl sulfone groups on the termini of the complementary PEG macromers. Thus, the crosslinking density of the hydrogel network could be tuned by uncaging with light which directly translated into differential patterns of hydrogel stiffness. Using this approach, user-defined stiffness patterns in a range of soft tissue microenvironments (i.e. between 3-8 kPa) were obtained and shown to influence the migratory behavior of primary human mesenchymal stem cells (hMSC). Stiffness gradients in the higher range (5.5-8 kPa) were able to elicit durotaxis towards the more densely crosslinked regions, whereas those in the lower range (3-5.5 kPa) showed no significant directional preference in hMSC migration. Our patterning tool should be useful for the manipulation of cell fate in various other contexts.