Microfluidic engineering of artificial stem cell niches

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 [...]

Hydrogel Microfluidics to Control Stem Cell Fate

Steffen Cosson

Biomolecular signaling is of utmost importance in governing many biological processes such as morphogenesis during tissue development where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion and in the context of a soft and hydrated microenvironment. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro- scale is far from ideal due to issues related to medium evaporation, limited space for cell growth, shear stress and a lack of cell-instructive microenvironments that might adversely impact cell-fate. As a result, microfluidic cell culture systems are not yet suitable to unravel complex multicellular phenomena. This may explain why they are not yet widely used in biology laboratories. Consequently, the overall goal of this thesis was to overcome this technology gap by developing next generation microfluidics through a combination of microfabrication and smart biomaterials. A special emphasis was placed on decoupling biomolecule presentation at micro-scale from macro-scale cell culture in order to enable long-term stem cell culture within user-friendly multiwell plate formats. First, a novel microfluidic concept was invented to rapidly immobilize linear protein gradients on the surface of poly(ethylene glycol) (PEG)-based hydrogels whose biophysical properties are reminiscent of natural extracellular matrices. This method allows efficient capture of steady-state gradients of tagged proteins (e.g. using Fc or biotin tags) in just a few minutes on engineered hydrogels that display the corresponding auxiliary proteins (e.g. ProteinA or NeutrAvidin). The selectivity and orthogonality of the chosen binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is impossible using existing platforms. After patterning, the microfluidic chip can be readily removed from the gel for cell culture applications. Using quantitative single-cell time-lapse microscopy, this platform was validated here by probing the effect of fibronectin concentration on the directionality and speed of cell migration. Next, the resolution, flexibility and throughput of the protein patterning on hydrogels was substantially enhanced by the introduction of hydrodynamic flow focusing, that is, the generation of user-defined patterns by spatially controlled step- wise deposition of biomolecules. To this end, a microfluidic device was conceived that enabled the generation of arrays of parallel and crossed overlapping gradients. Application of the platform to generate gradients of immobilized leukemia inhibitory factor (LIF) showed an influence of the LIF concentration on ESC self-renewal. This platform should be useful to systematically probe the effect of biomolecule dose, singly or in combinations, on stem cell behavior in vitro; however, it lacks the ability to dynamically vary the presentation of biomolecules that might be crucial to control stem cell fate towards establishing functional in vitro tissue models. To address this limitation, in the last part of the thesis work a hydrogel-based microfluidic chip was developed that decouples macro-scale cell culture on the gel surface from the precise spatiotemporal biomolecule delivery at the micro-scale, i.e. through the gel layer. The hydrogel chip, used here as a simple insert that is compatible with conventional multiwell plate formats, was optimized to support long- term ESC maintenance in both adherent format and as uniformly sized ESC aggregates termed embryoid bodies. The platform was successfully used for the spatially controlled neuronal commitment of ESC via delivery of gradients of the morphogen retinoic acid. Taken together, in this thesis several innovative microengineered cell culture platforms are presented which enable, perhaps for the first time, the long-term culture and manipulation of stem cell fate at the micro-scale. These systems should be useful to systematically probe in vitro the effect of biomolecule dose and delivery dynamics on stem cell behavior, ultimately facilitating the development of complex in vitro tissue models.

EPFL2012

Stem cell niche engineering through photopatterning and microfluidics

Laura Kolb

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.

EPFL2016

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

Stem cell niche engineering through photopatterning and microfluidics

Laura Kolb

EPFL2016

Photopatterning of Synthetic Hydrogels

Katarzyna Mosiewicz

EPFL2012

Hydrogel Microfluidics to Control Stem Cell Fate

Steffen Cosson

EPFL2012