Stem cell niche engineering through photopatterning and microfluidics

The application of stem cells in drug screening and regenerative therapy has led to important advances in basic biology and biomedicine. Such strategies require high cell numbers and the efficient maturation into faithful functional organ or tissue units. An important aspect to gain control over stem cell fate is the recapitulation of their natural instructive extracellular matrix (ECM). In vivo, stem cells reside in microenvironments, termed niches, providing distinct, tissue-specific signals. In a complex interplay, the fate decisions are tightly regulated by various parameters including biophysical and biochemical signals. Biomaterial science interfaced with sophisticated bioengineering techniques has provided powerful tools to recreate stem cell niches in vitro. However, major challenges for the efficient stem cell expansion and differentiation still have not been sufficiently addressed. In this thesis, platforms have been developed to help to reconstruct this dynamic and niche-specific signaling panoply. In one approach, a hydrogel crosslinking scheme based on a Michael-type addition reaction was combined with photosensitive caging molecules. By masking thiol moieties of a poly(ethylene glycol) (PEG) macromer with photolabile groups, the covalent crosslinking with vinylsulfone groups of complementary PEG macromers was spatiotemporally controlled by light illumination. By the generation of precisely controlled stiffness gradients, we were able to spatially direct human mesenchymal stem cell (hMSC) migration. Along the same lines, light-controlled uncaging was applied to tether biomolecules into a hydrogel matrix. PEG macromers were covalently functionalized with a peptide substrate of activated transglutaminase factor XIII (FXIIIa). This allowed for any desired ECM-derived peptide or protein fused to the counter-reactive peptide substrate to be enzymatically tethered into the hydrogel network. Photoprotection of the hydrogel-bound peptide substrate enabled exquisite dynamic control of biomolecule distribution. The patterning platform was exemplified by the guided three-dimensional (3D) invasion of encapsulated hMSCs into the hydrogel matrix. The second part of this work addressed the challenge of dissecting the vast molecular signaling networks that influence stem cell behavior. A droplet microfluidic-based screening platform was developed to study the effect of pivotal niche proteins in unparalleled combinatorial throughput via micron-sized hydrogel beads termed microniches. Randomized compartmentalization of stable inducible mammalian cell lines into microniches translated into myriads of combinations of secreted proteins tethered to the hydrogel matrix. Selected microniches with a desired phenotypic effect were analyzed for the protein combinations by targeted genotypic analysis of the underlying encapsulated stable mammalian lines. Positive instructive effects of microniches could be demonstrated on a model system of engineered HEK cells and are currently under evaluation for mesendodermal differentiation of a human embryonic stem cell (hESC) reporter cell line. The novel light-responsive materials as well as microfluidic-based screening approach presented here strive for a near-physiological niche signaling architecture. These tools can help to fully exploit the stem cell potential and accelerate the translation of stem cell-based applications from the laboratory towards the clinic.

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

Microfluidic engineering of artificial stem cell niches

Simone Allazetta

Stem cells play a key role in a wide range of biological processes, in large part due to their ability to self-renew or differentiate into specialized cell types in response to various biological cues. In vivo, stem cells reside in a complex microenvironment, termed niche, that regulates cell fate through intricate combinations of biophysical and biochemical factors. To recapitulate some of these crucial interactions ex vivo and to facilitate a quicker transition to clinical and pharmaceutical applications of stem cells, numerous technologies have been developed in recent years to better control and manipulate the cellular microenvironment. In particular, combining synthetic hydrogels having controllable mechanical and biochemical signaling with microfluidic technologies that can automatically and precisely handle fluids results in unprecedented control over in vitro microenvironmental conditions. In this thesis, novel microfluidic approaches were developed to engineer stem cell niches presenting defined and modular cell-instructive physicochemical cues. In a first approach, a novel platform based on computer-controlled hydrodynamic flow focusing was developed in order to tether steady-state gradients of tagged proteins, using Fc or biotin tags, onto the surface of poly(ethylene glycol) (PEG)-based hydrogels displaying selective capturing proteins (ProteinA or NeutrAvidin). This versatile patterning strategy permitted the generation of complex biomolecule gradients, with fine control over the patterning resolution and shape. Furthermore, the chosen binding schemes enabled parallel and orthogonal gradients of multiple proteins to be formed. As a proof-of-principle, we employed this technology to assess the influence of immobilized leukemia inhibitor factor (LIF) concentration on mouse embryonic stem cell self-renewal. While this technology allowed biomolecule dose effect on stem cell behavior to be investigated, it was limited in its ability to modulate multiple microenvironmental factors simultaneously. To address this limitation, droplet-based microfluidic technology was adopted which allows perturbing microenvironmental conditions in a combinatorial fashion and with nearly unmatched precision and throughput. To this end, microfluidic chips were fabricated for the generation of PEG-based microgels with precisely controlled dimensions and physico-chemical properties. First, we developed a versatile biofunctionalization technique for tethering biomolecules of interest to the reactive PEG microgels. Then, selective peptide- and protein-modified formulations were tested for their ability to promote adhesion and proliferation of various stem cell types in a bioreactor-based suspension culture. Next, programmable modulation of the flows was adopted to generate microgels with varying elasticity, biochemical ligands or both. The different microgel properties were encoded with specific fluorescent markers such that the microenvironment properties could be readily identified via microscopy or flow cytometry. Controlling syringe pumps through computer programming allowed us to generate up to 100 populations of microgels with different elasticities and bioactive ligand concentrations in a single experiment. Thousands of compositionally distinct microgels were then analyzed by the intensity levels of two fluorescent moieties incorporated in the hydrogel network. As a proof-of-principle, we demonstrated that this technology can be adopted to [...]

EPFL2015

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