Mimicking Sarcolemmal Damage In vitro: A 3D Skeletal Muscle Model for Drug Testing in Duchenne Muscular Dystrophy
Ainoa Tejedera Villafranca, Biosensors for bioengineering group
Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disease diagnosed in childhood. It is a progressive and wasting disease, characterized by a degeneration of skeletal and cardiac muscles caused by the lack of dystrophin protein. The absence of this structural protein leads to the fragility of the sarcolemma, and muscle fibers are damaged during their contraction. To date, there is no cure available for patients, even though there are several molecules in drug development. However, due to the well-known limitations of preclinical research, the success rate of drugs remains low. In this work, intending to accelerate drug discovery for DMD, we developed a patient-derived functional 3D model of DMD. By using a 3D-printed casting mold, we encapsulated muscle progenitor cells in a fibrin-composite matrix. This platform incorporates two flexible T-shaped pillars that serve as anchoring points and provide continuous tension to the tissue, thus allowing the orientation of the muscle fibers. The skeletal muscle tissues expressed mature myogenic markers and showed functional phenotypes as they responded to electrical pulse stimulation (EPS) by contracting. We observed that DMD muscle tissues, after continuous contractile regimes, reproduced the loss of myotube integrity that is observed in dystrophinopathies due to the sarcolemmal instability. DMD but not healthy tissues showed functional phenotypes caused by the induced sarcolemmal damage, such as tetanic fatigue. Finally, the applicability of this DMD model in evaluating therapeutic compounds was explored. Specifically, we investigate the effect of utrophin up-regulators on functional outcomes of the model, thus identifying potential candidates for the treatment of DMD. Taking all these considerations together, our results show that bioengineered 3D skeletal muscle technology has great potential to be especially valuable in the context of current and future discovery and development of drugs to treat DMD and other neuromuscular disorders.
Dissecting early nephron patterning and segmentation in kidney organoids derived from hPSCs
Zarina Nauryzgaliyeva , Pluripotency for organ regeneration Group
The formation and maturation of organs during development is a complex, yet beautifully orchestrated process. Embryonic cells have a unique capacity to self-organize within the forming tissue, where morphogenetic movements have been shown to facilitate tissue organization and subsequent organ formation. In kidney organogenesis, the mature organ arises from crucial reciprocal interactions between the ureteric bud (UB) and metanephric mesenchyme (MM), which give rise to the collecting duct and nephron, respectively. The development of mature nephrons during kidney organogenesis is a dynamic process so far studied taking advantage of in vivo models. Accumalative findings in mice have shown that the MM undergoes mesenchymal to epithelial transition (MET), giving rise to epithelial renal vesicles (RVs) that further undergo structural changes and shift towards comma shaped and s-shaped bodies (CSBs/SSBs), which eventually develop into nephron like structures. Those studies have helped identify Wnt/b-catenin and Notch signalling pathways as key players in nephron patterning and segmentation (proximal, medial, distal segments).
At the same time, tissue morphogenesis is largely a biomechanical process, resulting from constant movements of cells, changes in forms of developing segments and forces generated therein. The biomechanical dynamics occurring during RV emergence and further nephron patterning are yet to be explored in the human context in real time. If these biomechanical processes are interconnected with mechanical signals remains an open question in the field. The answer to these questions may have an important impact for understanding nephron formation, and conversely, disease-related phenotypes due to mutations in genes orchestrating RV patterning and segmentation as occurs in congenital defects of the kidney and the urinary tract (CAKUT disease).
Here, we aim to use human pluripotent stem cell (hPSCs) derived kidney organoids to gain fundamental understanding of early nephron patterning and segmentation by mapping force transmission between cells and their extracellular matrix (ECM) and evaluating their co-evolution during renal fate specification and differentiation.
hPSCs are guided towards the renal fate on compliant PDMS hydrogels with controlled rigidities (mimicking embryonic microenvironment) in a 2D culture system. PDMS hydrogels between 3 kPa (soft) and 18 kPa (rigid) are generated by adapting the compositional ratio of PDMS components and are further functionalized and decorated with fibronectin. Using this system, we have started to spatiotemporally characterise early steps of nephrogenesis by immunofluorescence and confocal analysis, time-lapse imaging, and traction force microscopy (TFM). These analyses are nowadays conducted during RV emergence prior proximal-distal RV polarization and formation of the nephron-like segments.
The current techniques will permit quantitative and qualitative observations of multicellular behaviours at key stages of 2D renal differentiation. Furthermore, this system will allow us to spatiotemporally map cell-cell and cell-ECM forces and evaluate their evolution throughout renal fate specification with the final aim to decouple mechano-related processes sustaining nephron formation from classical biochemical signalling.