Cell-Instructive Microgels with Tailor-Made Physicochemical Properties

A microfluidic in vitro cell encapsulation platform to systematically test the effects of microenvironmental parameters on cell fate in 3D is developed. Multiple cell types including fibroblasts, embryonic stem cells, and cancer cells are incorporated in enzymatically cross-linked poly(ethylene glycol)-based microgels having defined and tunable mechanical and biochemical properties. Furthermore, different approaches to prevent cell "escape" from the microcapsules are explored and shown to substantially enhance the potential of this technology. Finally, coencapsulation of microgels within nondegradable gels allows cell viability, proliferation, and morphology to be studied in different microenvironmental conditions up to two weeks in culture.

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

Induction of cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling

Freddy Radtke

Embryonic stem cells represent an attractive source of cardiomyocytes for cell-replacement therapies. However, before embryonic stem cells can be successfully used for the treatment of cardiac diseases, the precise molecular mechanisms that underlie their cardiogenic differentiation must be identified. A network of intrinsic and extrinsic factors regulates embryonic stem cell self-renewal and differentiation into a variety of different cell lineages. Here, we show that Notch signaling takes place in some but not all embryonic stem cells and that the Notch pathway is shut down during the course of differentiation concomitantly with downregulation of Notch receptor and ligand expression. Moreover, gain- and loss-of-function experiments for Notch signaling components show that this pathway is a crucial regulator of cardiomyocyte differentiation within ES cells. Differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch1 receptor, whereas endogenous Notch signaling promotes differentiation of ES cells into the neuronal lineage. We conclude that Notch signaling influences the cell fate decision between mesodermal and the neuroectodermal cell fates during embryonic stem cell differentiation. These findings should help to optimize the production of specific cell types via modulation of the Notch pathways and, in particular, to improve the production of embryonic stem cell-derived cardiomyocytes.

2006

Development of bioactive hydrogel capsules for the 3D expansion of pluripotent stem cells in bioreactors

Matthias Lütolf, Yoji Tabata

Pluripotent stem cells hold great promise for many pharmaceutical and therapeutic applications. However, the lack of scalable methodologies to expand these cells to clinically relevant numbers is a major roadblock in realizing their full potential. To address this problem, we report here a scalable approach for the expansion of pluripotent stem cells within bioactive hydrogel capsules in stirred bioreactors. To achieve rapid crosslinking of cellular microenvironments with tuneable, cell-instructive functionality, we combined calcium-mediated alginate (CaAlg) complexation with crosslinking of poly-(ethylene glycol) (PEG) macromers via a Michael-type addition. The resulting hybrid networks have been shown to have very good handling properties and can be readily decorated with biologically active signals such as integrin ligands or Cadherin-based motifs to influence the fate of mouse induced pluripotent stem (iPS) cells. Air-driven co-axial extrusion was used to reproducibly generate gel microcapsules in high-throughput. Furthermore, the gel capsules can be enveloped in a poly(L-lysine) shell to control swelling or molecular permeability independently of the gel composition. iPS cells entrapped within such capsules expanded with limited commitment to the endodermal lineage. Functionalization of gels with an appropriate density of Arg-Gly-Asp (RGD) ligands further increased the iPS cell expansion rate and reduced the spontaneous differentiation. Therefore, the combination of micro-scale instruction of cell fate by an engineered microenvironment and macro-scale cell manipulation in bioreactors opens up exciting opportunities for stem cell-based applications.

Royal Soc Chemistry2014

Stem cell niche engineering through photopatterning and microfluidics

Laura Kolb

EPFL2016