Bioengineering stem cell niches in 2D and 3D

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.

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

Bioengineering the Human Muscle Stem-Cell Niche for Therapeutic Applications

Omid Mashinchian

In spite of decades of research, no feasible method for obtaining sufficient numbers of uncommitted muscle stem cells (MuSCs) for therapy of degenerative muscle diseases exists. One of the most fundamental problems associated with stem cell therapy of muscle is that removal of MuSCs from their tissue microenvironment and expansion in conventional culture induces their terminal commitment to myogenic differentiation. This in-vitro loss of stemness impairs the long-term engraftment potential of MuSCs and renders them unsuitable for stem cell therapy. In another disease relevant context, aging, dramatic changes in the functionality of MuSCs have been shown to depend on an altered composition of their microenvironment. Thus, muscle progenitors are fundamentally dependent on their local environment, commonly known as the âstem cell niche", and a better understanding of its role in guiding stem cell function is highly relevant for the development of therapies for aging and muscle diseases. The muscle stem cell niche is composed of diverse cellular and acellular elements, including different supportive cell types, growth factors and extracellular matrix, and as outlined above, is critical for maintaining healthy stem cell characteristics such as the capacity for self-renewal, commitment, and differentiation. Using molecular biology approaches, we decided to interrogate (I) the role of the cellular environment on stem cell commitment, (II) to test whether, in a physiological context, the systemic circulation has niche-independent effects on MuSC stemness, and (III) to study a population of MuSC-supportive cells that is affected by the aging process. In the first study of this thesis, we have successfully developed an organoid-like-approach for the scalable derivation of uncommitted MuSCs from human induced pluripotent stem cells (iPSCs) in a biologically faithful cellular 3D-environment in suspension embryoids. To this end, we employed human iPSCs and a spectrum of immortalized cell lines to screen for 3D-aggregation conditions promoting mesoderm formation and subsequent specification to the myogenic lineage without the parallel upregulation of myogenic commitment markers. Compared to myogenic cells derived from adult human skeletal muscle, this niche-mimetic embryoid derived progenitors display significantly enhanced engraftment into the muscle stem cell position peripheral to muscle fibers, and restoration of dystrophin expression when transplanted into muscles of a mouse model of Duchenne muscular dystrophy. In a second study, we present a novel method for encapsulation of human muscle progenitors in highly diffusible polyethersulfone hollow fiber capsules to identify highly specific "transcriptional-signature" induced by systemic aging in-vivo. This technique allows to study the systemic circulation in health, disease and aging at an unprecedented level in human cell types of choice. Finally, in our third study, we investigated the cellular cross-talk between muscle stem cells and non-myogenic niche-based cell type, called fibro/adipogenic progenitors (FAPs). Interestingly, the support-function and the cross-talk with stem cells are dramatically impaired in aged FAPs. We demonstrate that this mechanism can be targeted to rejuvenate myogenesis. Taken together, "mimicking" the physicochemical-interactions of MuSCs with their niche-resident cell types, represents a powerful opportunity to manipulate MuSC function in aging and disease.

EPFL2019

Muscle stem cell's in vitro bipolar niche

Adrien de Tonnac

In the domain of regenerative medicine research there are many fronts aimed at increasing our understanding of induced pluripotent, embryonic and adult stem cells. Recently a new front has appeared, stem cell niches, the microenvironment that is a crucial component in the regulation of stem cells. New in vitro platforms are continually being created to answer the multitude of biological questions that surround stem cell niches. The transition from in vitro to in vivo is a heavy setback in cellular research due to a huge gap between the two conditions, which can only be reduced if the in vitro conditions better mimic those found in vivo. This project has reduced the gap and has the potential to continue minimizing the differences between the two conditions in the field of muscle stem cell research. Through a combination of the forefront techniques in biological research, we were able to create a new innovative bioengineered platform, a bipolar hydrogel microenvironment. First, we took advantage of polyethylene glycol (PEG) hydrogels, which are highly hydrophilic polymer networks that have properties that mimic physiologic tissue, including high water content and low Young's Modulus. Microfabrication techniques allowed us to topographically pattern the hydrogel at micro-dimensions resembling the native cell environment. Utilizing microfluidics and micro-printing techniques, the three-dimensional hydrogel surface was also patterned with tethered niche proteins found in the in vivo muscle stem cell niche. The swelling properties of the PEG hydrogel were considered as an advantage and controlled to form niche gaps with physical properties that were tuned to correspond to those found in vivo. This new complex and controlled surface for cells to be cultured on is a tool, which will allow biologists to probe new questions and gain insight into the regulatory networks that control cell fate